Magnesium chelatase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a magnesium chelatase subunit. The invention also relates to the construction of a chimeric gene encoding all or a substantial portion of the magnesium chelatase subunit, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the magnesium chelatase subunit in a transformed host cell.

[0001] This application claims the benefit of U.S. Provisional Application No. 60/137,461, filed Jun. 4, 1999.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding magnesium chelatase subunit in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Magnesium chelatase catalyzes the insertion of the magnesium cation (Mg²⁺) into protoporphyrin IX, the branchpoint in the tetrapyrrole biosynthetic pathways leading to (bacterio)chlorophyll synthesis. In photosynthetic bacteria, magnesium chelatase activity requires three different subunits encoded by the genes bchD, bchH and bchI (Willows, R. D. and Beale, S. I., (1998) J. Biol. Chem. 273:34206-34213). It has been proposed that the BchH subunit initially forms a complex with protoporphyrin IX while the Bch I and BchD subunits form a complex in an ATP-dependent activation step. The BchI-BchD complex then inserts the magnesium cation into the BchH-bound protoporphyrin IX in an ATP-dependent reaction (Willows, R. D. and Beale, S. I. supra).

[0004] Similarly in higher plants, three distinct proteins, CHLD, CHLH, and CHLI, encoded by the genes ChID, ChIH and ChII respectively (Papenbrock J. et al., (1997) Plant J. 12:981-990) are required for magnesium chelatase activity. They share significant sequence similarity with their bacterial counterparts, further suggesting that the mechanism of magnesium chelation proceeds more or less similarly in plants and bacteria (Guo, R. et al., (1998) Plant Physiol. 116:605-615).

[0005] Since magnesium chelatase is an enzyme specific for chlorophyll synthesis, it presents a potential target for discovery and development of herbicides nontoxic to man and animals. Isolation of more genes encoding magnesium chelatase subunits provides a wider array of possible targets, thereby increasing the chances of successfully identifying promising herbicide candidates.

SUMMARY OF THE INVENTION

[0006] The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 38, 42, and 48; (b) a second nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 10, and 30; (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26; (d) a fourth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 20, and 34; (e) a fifth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24; (f) a sixth nucleotide sequence encoding a polypeptide of at least 130 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28; (g) a seventh nucleotide sequence encoding a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:32; (h) an eighth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:44; (i) a ninth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22; (j) a tenth nucleotide sequence encoding a polypeptide of at least 380 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; (k) an eleventh nucleotide sequence encoding a polypeptide of at least 400 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:50; (l) a twelfth nucleotide sequence encoding a polypeptide of at least 400 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:4; (m) a thirteenth nucleotide sequence encoding a polypeptide of at least 750 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:12; (n) a fourteenth nucleotide sequence encoding a polypeptide of at least 1110 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:36; and (o) a fifteenth nucleotide sequence comprising the complement of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), or (n).

[0007] In a second embodiment, it is preferred that the isolated polynucleotide of the claimed invention comprises a nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 48, and 50.

[0008] In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 and the complement of such nucleotide sequences.

[0009] In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.

[0010] In a fifth embodiment, the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

[0011] In a sixth embodiment, the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.

[0012] In a seventh embodiment, the invention concerns a magnesium chelatase subunit polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 38, 42, and 48; (b) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 10, and 30; (c) a polypeptide of at least 100 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26; (d) a polypeptide of at least 100 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 20, and 34; (e) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24; (f) a polypeptide of at least 130 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28; (g) a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:32; (h) a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:44; (i) a polypeptide of at least 250 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22; (j) a polypeptide of at least 380 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; (k) a polypeptide of at least 400 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:50; (l) a polypeptide of at least 400 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:4; (m) a polypeptide of at least 750 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:12; and (n) a polypeptide of at least 1110 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:36.

[0013] In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a magnesium chelatase subunit polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the chimeric gene into a host cell; (c) measuring the level of the magnesium chelatase subunit polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the magnesium chelatase subunit polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the magnesium chelatase subunit polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.

[0014] In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a magnesium chelatase subunit polypeptide, preferably a plant magnesium chelatase subunit polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a magnesium chelatase subunit amino acid sequence.

[0015] In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a magnesium chelatase subunit polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

[0016] In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or an isolated polypeptide of the present invention.

[0017] In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or a construct of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the magnesium chelatase subunit polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.

[0018] In a thirteenth embodiment, this invention relates to a method of altering the level of expression of a magnesium chelatase subunit in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the magnesium chelatase subunit in the transformed host cell.

[0019] A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a magnesium chelatase subunit, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a magnesium chelatase subunit polypeptide, operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the encoded magnesium chelatase subunit in the transformed host cell; (c) optionally purifying the magnesium chelatase subunit polypeptide expressed by the transformed host cell; (d) treating the magnesium chelatase subunit polypeptide with a compound to be tested; and (e) comparing the activity of the magnesium chelatase subunit polypeptide that has been treated with a test compound to the activity of an untreated magnesium chelatase subunit polypeptide, thereby selecting compounds with potential for inhibitory activity.

[0020] Another embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a magnesium chelatase subunit, the method comprising the steps of: (a) transforming a host cell or plant with a chimeric gene comprising a nucleic acid fragment encoding a magnesium chelatase subunit polypeptide, operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell or plant under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the magnesium chelatase subunit encoded by the operably linked nucleic acid fragment in the transformed host cell or plant; (c) treating the transformed host cell or plant with a compound to be tested; and (d) comparing the viability of the transformed host cell or plant that has been treated with a test compound to the viability of an untreated transformed host cell or plant, thereby selecting compounds with potential for inhibitory activity. Methods for determining viability of cells and plants are well-known to those of ordinary skill in the art.

[0021] A further embodiment of the instant invention is a method for conferring, to a host cell or plant, resistance to herbicidal compounds acting on magnesium chelatase, the method comprising the steps of: (a) transforming a host cell or plant with a chimeric gene comprising a nucleic acid fragment encoding a magnesium chelatase subunit polypeptide, operably linked to at least one suitable regulatory sequence; and (b) growing the transformed host cell or plant under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the magnesium chelatase subunit encoded by the operably linked nucleic acid fragment in the transformed host cell or plant, and results further in resistance of the transformed host cell or plant to herbicidal compounds acting on magnesium chelatase.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0022] The invention can be more fully understood from the following detailed description, the accompanying drawings and the Sequence Listing which form a part of this application.

[0023]FIG. 1 depicts the amino acid sequence alignment of the CHLD proteins encoded by the nucleotide sequences derived from a contig assembled from corn clone p0005.cbmff04r and PCR product (SEQ ID NO:4), and soybean clone sdp4c.pk022.h18 (SEQ ID NO:12), and the CHLD protein from Pisum sativum (NCBI GI No. 3913218; SEQ ID NO:51). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.

[0024]FIG. 2 depicts the amino acid sequence alignment of CHLI proteins encoded by the nucleotide sequences derived from corn clone csc1c.pk004.p11 (SEQ ID NO:44) and rice clone rls48.pk0001.h1 (SEQ ID NO:50), and the CHLI protein from Glycine max (NCBI GI No. 3334150; SEQ ID NO:52). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.

[0025] Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”). Nucleotide SEQ ID NOs:1, 5, 9, 13, 15, 19, 23, 33, 37, 41, 45, and 47 correspond to nucleotide SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, and 21, respectively, presented in U.S. Provisional Application No. 60/137,461, filed Jun. 4, 1999. Amino acid SEQ ID NOs:2, 6, 10, 14, 16, 20, 24, 34, 38, 42, 46, and 48 correspond to amino acid SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, and 22, respectively, presented in U.S. Provisional Application No. 60/137,461, filed Jun. 4, 1999. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. TABLE 1 Magnesium Chelatase Subunits Protein (Plant Clone SEQ ID NO: Source) Designation Status (Nucleotide) (Amino Acid) CHLD p0005.cbmff04r EST 1 2 (Corn) CHLD Contig of: CGS 3 4 (Corn) p0005.cbmff04r (FIS) PCR product CHLD rsl1n.pk001.j9 EST 5 6 (Rice) CHLD rsl1n.pk001.j9 FIS 7 8 (Rice) CHLD (Soy- sdp4c.pk022.h18 EST 9 10 bean) CHLD (Soy- sdp4c.pk022.h18 CGS 11 12 bean) (FIS) CHLD (Soy- ses4d.pk0043.c6 EST 13 14 bean) CHLD wr1.pk0064.e2 EST 15 16 (Wheat) CHLD wr1.pk0064.e2 FIS 17 18 (Wheat) CHLH cdt1c.pk001.o1 EST 19 20 (Corn) CHLH cdt1c.pk001.o1 FIS 21 22 (Corn) CHLH chp2.pk0007.d5 EST 23 24 (Corn) CHLH chp2.pk0014.h1 EST 25 26 (Corn) CHLH p0019.clwaa84r EST 27 28 (Corn) CHLH p0088.clrih26r EST 29 30 (Corn) CHLH p0110.cgsmo74r EST 31 32 (Corn) CHLH rlr2.pk0018.e9 EST 33 34 (Rice) CHLH rlr2.pk0018.e9 FIS 35 36 (Rice) CHLH wdk4c.pk005.f24 EST 37 38 (Wheat) CHLH wdk4c.pk005.f24 FIS 39 40 (Wheat) CHLI csc1c.pk004.p11 EST 41 42 (Corn) CHLI csc1c.pk004.p11 CGS 43 44 (Corn) (FIS) CHLI (Rice) rlr72.pk0014.f9 EST 45 46 CHLI (Rice) rls48.pk0001.h1 EST 47 48 CHLI (Rice) rls48.pk0001.h1 CGS 49 50 (FIS)

[0026] SEQ ID NOs:53 and 54 are oligonucleotide primers used in PCR amplification of 5′ end of the cDNA represented in clone p0005.cbmff04r.

[0027] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0028] In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49, or the complement of such sequences.

[0029] The term “isolated polynucleotide” refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.

[0030] The term “recombinant” means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.

[0031] As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

[0032] As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. The terms “substantially similar” and “corresponding substantially” are used interchangeably herein.

[0033] Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

[0034] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a magnesium chelatase subunit polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; introducing the isolated polynucleotide or the chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.

[0035] Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6× SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2× SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2× SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2× SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1× SSC, 0.1% SDS at 65° C.

[0036] Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 or 130 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250, 380, 400, 750 or 1110 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0037] A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0038] “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0039] “Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0040] “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0041] “Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0042] “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

[0043] “Translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

[0044] “3′ Non-coding sequences” refers to nucleotide sequences located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0045] “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and can be translated into polypeptides by the cell. “cDNA” refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I. “Sense RNA” refers to an RNA transcript that includes the mRNA and can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

[0046] The term “operably linked” refers to the association of two or more nucleic acid fragments so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0047] The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. “Expression” may also refer to the translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

[0048] A “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.

[0049] “Altered levels” or “altered expression” refer to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

[0050] “Null mutant” refers to a host cell which either lacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.

[0051] “Mature protein” or the term “mature” when used in describing a protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor protein” or the term “precursor” when used in describing a protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

[0052] A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

[0053] “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0054] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

[0055] “PCR” or “polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0056] The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 38, 42, and 48; (b) a second nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 10, and 30; (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26; (d) a fourth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 20, and 34; (e) a fifth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24; (f) a sixth nucleotide sequence encoding a polypeptide of at least 130 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28; (g) a seventh nucleotide sequence encoding a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:32; (h) an eighth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:44; (i) a ninth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22; (j) a tenth nucleotide sequence encoding a polypeptide of at least 380 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; (k) an eleventh nucleotide sequence encoding a polypeptide of at least 400 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:50; (l) a twelfth nucleotide sequence encoding a polypeptide of at least 400 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:4; (m) a thirteenth nucleotide sequence encoding a polypeptide of at least 750 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:12; (n) a fourteenth nucleotide sequence encoding a polypeptide of at least 1110 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:36; and (o) a fifteenth nucleotide sequence comprising the complement of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), or (n).

[0057] Preferably, the nucleotide sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49, that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 48, and 50.

[0058] Nucleic acid fragments encoding at least a substantial portion of several magnesium chelatase subunits have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

[0059] For example, genes encoding other magnesium chelatase subunits, either as cDNAs or genomic DNAs, could be isolated directly by using all or a substantial portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, entire sequence(s) can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

[0060] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.

[0061] The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a magnesium chelatase subunit polypeptide, preferably a substantial portion of a plant magnesium chelatase subunit polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a magnesium chelatase subunit polypeptide.

[0062] Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing substantial portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0063] In another embodiment, this invention concerns viruses and host cells comprising either the chimeric genes of the invention as described herein or an isolated polynucleotide of the invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.

[0064] As was noted above, the nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of magnesium cation (Mg²⁺) insertion into protoporphyrin IX in those cells, consequently leading to changes in the level of chlorophyll pigmentation in those cells. Another effect would be changes in the level of resistance of those cells to herbicidal compounds acting on magnesium chelatase; overexpression of magnesium chelatase subunits may confer resistance to herbicidal compounds acting on magnesium chelatase. The nucleic acid fragments of the instant invention may also be used for overexpression in bacterial or yeast hosts, thereby efficiently producing large amounts of the encoded polypeptides which could then be used for screening different compounds for potential herbicidal activity. Host cells (e.g., plant, cyanobacteria) overexpressing magnesium chelatase may also be used directly for screening different compounds for potential herbicidal activity by exposing said host cell directly to the compound being tested.

[0065] Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

[0066] Plasmid vectors comprising the instant isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0067] For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate their secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.

[0068] It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

[0069] Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of a specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

[0070] The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

[0071] In another embodiment, the present invention concerns a magnesium chelatase subunit polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 38, 42, and 48; (b) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 10, and 30; (c) a polypeptide of at least 100 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26; (d) a polypeptide of at least 100 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 20, and 34; (e) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24; (f) a polypeptide of at least 130 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28; (g) a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:32; (h) a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:44; (i) a polypeptide of at least 250 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22; (j) a polypeptide of at least 380 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; (k) a polypeptide of at least 400 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:50; (l) a polypeptide of at least 400 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:4; (m) a polypeptide of at least 750 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:12; and (n) a polypeptide of at least 1110 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:36.

[0072] The instant polypeptides (or substantial portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded magnesium chelatase subunit. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 8).

[0073] Additionally, the instant polypeptides can be used as a target to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptides described herein catalyze various steps in magnesium chelation of protoporphyrin IX, en route to chlorophyll production. Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition of plant growth. Thus, the instant polypeptides could be appropriate for new herbicide discovery and design.

[0074] All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0075] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0076] Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0077] In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0078] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

[0079] Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptide. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptide can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

EXAMPLES

[0080] The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0081] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0082] cDNA libraries representing mRNAs from various corn, rice, soybean and wheat tissues were prepared. The characteristics of the libraries are described below. Corn developmental stages are explained in the publication “How a corn plant develops” from the Iowa State University Coop. Ext. Service Special Report No. 48 reprinted June 1993. TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat Library Tissue Clone cdt1c Corn Developing Tassel cdt1c.pk001.o1 chp2 Corn (B73 and MK593) 11 Day Old Leaf chp2.pk0007.d5 Treated 24 Hours With Herbicides* chp2.pk0014.h1 csc1c Corn 20-Day-Old Seedling (Germination csc1c.pk004.p11 Cold Stress) p0005 Corn Immature Ear p0005.cbmff04r p0019 Corn Green Leaves (V5-7) After Mechanical p0019.clwaa84r Wounding for 1 Hour p0088 Corn Leaf: Induced Resistance; Harvested p0088.clrih26r Prior To Spontaneous Lesion Formation; About One Month After Planting In Green House**;*** p0110 Corn (Stages V3/V4) Leaf Tissue Minus p0110.cgsmo74r Midrib Harvested 4 Hours, 24 Hours and 7 Days After Infil- tration With Salicylic Acid, Pooled** rlr2 Resistant Rice Leaf 15 Days After Germi- rlr2.pk0018.e9 nation, 2 Hours After Infection of Strain Magaporthe grisea 4360-R-62 (AVR2- YAMO) rlr72 Resistant Rice Leaf 15 Days After Germi- rlr72.pk0014.f9 nation, 72 Hours After Infection of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO) rls48 Susceptible Rice Leaf 15 Days After Germi- rls48.pk0001.h1 nation, 48 Hours After Infection of Strain Magaporthe grisea 4360-R-67 (AVR2-YAMO) rsl1n Rice 15-Day-Old Seedling** rsl1n.pk001.j9 sdp4c Soybean Developing Pod (10-12 mm) sdp4c.pk022.h18 ses4d Soybean Embryogenic Suspension 4 Days ses4d.pk0043.c6 After Subculture wdk4c Wheat Developing Kernel, 21 Days After wdk4c.pk005.f24 Anthesis wr1 Wheat Root From 7-Day-Old Seedling Light wr1.pk0064.e2 Grown #4,2′-[1,3]dioxolan]-6-yl)carbonyl]-3-hydroxy-2-cyclohexen-1-one S,S-dioxide; synthesis and methods of using this compound are described in WO 97/01550, incorporated herein by reference)

[0083] cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0084] cDNA clones encoding magnesium chelatase subunit were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as “pLog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Magnesium Chelatase Subunit CHLD

[0085] The BLASTX search using the EST sequences from clones rsl1n.pk001.j9, sdp4c.pk022.h18, ses4d.pk0043.c6 and wr1.pk0064.e2 revealed similarity of the proteins encoded by the cDNAs to magnesium chelatase subunit CHLD from Pisum sativum (NCBI GenBank Identifier No. 3913218). The BLAST results for each of these ESTs are shown in Table 3: TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous to Magnesium Chelatase Subunit CHLD Clone BLAST pLog Score (3913218) rsl1n.pk001.j9 68.70 sdp4c.pk022.h18 36.40 ses4d.pk0043.c6 21.52 wr1.pk0064.e2 61.00

[0086] The BLASTX search using the EST sequences from clone p0005.cbmff04r revealed similarity of the protein encoded by the cDNA to magnesium chelatase subunit CHLD from Nicotiana tabacum (NCBI GenBank Identifier No. 3913240) with a pLog score of 58.70.

[0087] The sequence of a substantial portion of the cDNA insert from clone p0005.cbmff04r is shown in SEQ ID NO:1; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:2. The sequence of a substantial portion of the cDNA insert from clone rsl1n.pk001.j9 is shown in SEQ ID NO:5; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:6. The sequence of a substantial portion of the cDNA insert from clone sdp4c.pk022.h18 is shown in SEQ ID NO:9; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:10. The sequence of a substantial portion of the cDNA insert from clone ses4d.pk0043.c6 is shown in SEQ ID NO:13; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:14. The sequence of a substantial portion of the cDNA insert from clone wr1.pk0064.e2 is shown in SEQ ID NO:15; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:16. BLAST scores and probabilities indicate that the instant nucleic acid fragments encode substantial portions of magnesium chelatase subunit CHLD.

[0088] The BLASTX search using the EST sequences from clones listed in Table 4 revealed similarity of the polypeptides encoded by the cDNAs to magnesium chelatase subunit CHLD from Hordeum vulgare (NCBI GenBank Identifier (GI) No. 6066383), Nicotiana tabacum (NCBI GI No. 3913240), and Pisum sativum (NCBI GI No. 3913218). Shown in Table 4 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”): TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous to Magnesium Chelatase Subunit CHLD BLAST Results Clone Status NCBI GI No. pLog Score Contig of: CGS 3913240 >254.00 p0005.cbmff04r (FIS) PCR product rsl1n.pk001.j9 FIS 6066383 >254.00 sdp4c.pk022.h18 CGS 3913218 >254.00 (FIS) wr1.pk0064.e2 FIS 6066383 103.00

[0089] In generating the PCR product whose sequence was used in a contig with the sequence of the entire insert in clone p0005.cbmff04r to generate the nucleotide sequence encoding the entire protein, the following oligonucleotides were used as primers: Cgs252-RIa: 5′ GGAAGCATAGCATGCAAACCAC 3′ SEQ ID NO:53 Cgs252-ROa: 5′ CACTTCAATGGGTGGAAGCATAG 3′ SEQ ID NO:54

[0090] Template DNA used in separate reactions was DNA from a cDNA library made from cDNA derived from corn developing kernel (embryo and endosperm) 10 days after pollination (library cbn10); DNA from a cDNA library made from cDNA derived from the root of 7-day-old corn seedling (library cr1); and DNA from a cDNA library made from cDNA derived from a corn embryo 20 days after pollination (library cho1c). The PCR reaction mix contained the following components, with GC polymerase mix, GC buffer, and GC melt obtained from Clonetech: GC Polymerase Mix, 50X 1 μL GC Buffer, 5X 10 μL GC Melt, 1 M 10 μL dNTPs, 2 mM each 5 μL Template DNA 1-5 μL Primer 1, 10 μM (SEQ ID NO:53) 1 μL Primer 2, 10 μM (SEQ ID NO:54) 1 μL Water to a total volume of 50 μL

[0091] PCR conditions were as follows:

[0092] 1 cycle of 94° C., 1 minute;

[0093] 10 cycles of

[0094] 94° C., 30 seconds,

[0095] 68° C., 30 seconds,

[0096] 72° C., 4 minutes,

[0097] with annealing temperature decreasing 0.5° C. per cycle to a final temperature of 63° C.;

[0098] 25 cycles of

[0099] 94° C., 30 seconds,

[0100] 63° C., 30 seconds,

[0101] 72° C., 4 minutes;

[0102] 1 cycle of 72° C., 7 minutes;

[0103] Hold at 15° C. until next step.

[0104] PCR products were subcloned using Topo TA Cloning kit (Invitrogen) and sequenced. Sequencing of PCR products yielded nucleotides 1-395 in SEQ ID NO:4 which overlaps with the sequence of the entire insert in clone p0005.cbmff04r. A contig was thus assembled, shown in SEQ ID NO:4.

[0105]FIG. 1 presents an alignment of the amino acid sequences set forth in SEQ ID NOs:4 and 12 and the Pisum sativum sequence (NCBI GI No. 3913218; SEQ ID NO:51). The data in Table 5 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:4 and 12 and the Pisum sativum sequence (NCBI GI No. 3913218; SEQ ID NO:51). TABLE 5 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Magnesium Chelatase Subunit CHLD Percent Identity to SEQ ID NO. NCBI GI No. 3913218; SEQ ID NO:51 4 78.0 12 85.1

[0106] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments, BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode all or a substantial portion of a magnesium chelatase subunit CHLD.

Example 4 Characterization of cDNA Clones Encoding Magnesium Chelatase Subunit CHLH

[0107] The BLASTX search using the EST sequences from clones cdt1c.pk001.o1 and chp2.pk0007.d5 revealed similarity of the proteins encoded by the cDNAs to magnesium chelatase subunit CHLH from Glycine max (NCBI GenBank Identifier (GI) No. 3059095) with pLog scores of 50.22 and 90.30, respectively. The BLASTX search using the EST sequences from clones rlr2.pk0018.e9 and wdk4c.pk005.f24 revealed similarity of the proteins encoded by the cDNAs to magnesium chelatase subunit CHLH from Hordeum vulgare (NCBI GI No. 2130042) with pLog scores of 60.70 and 13.05, respectively.

[0108] The sequence of a substantial portion of the cDNA insert from clone cdt1c.pk001.o1 is shown in SEQ ID NO:19; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:20. The sequence of a substantial portion of the cDNA insert from clone chp2.pk0007.d5 is shown in SEQ ID NO:23; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:24. The sequence of a substantial portion of the cDNA insert from clone rlr2.pk0018.e9 is shown in SEQ ID NO:33; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:34. The sequence of a substantial portion of the cDNA insert from clone wdk4c.pk005.f24 is shown in SEQ ID NO:37; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:38. BLAST scores and probabilities indicate that the instant nucleic acid fragments encode substantial portions of magnesium chelatase subunit CHLH.

[0109] The BLASTX search using the EST sequences from clones listed in Table 6 revealed similarity of the polypeptides encoded by the cDNAs to magnesium chelatase subunit CHLH from Glycine max (NCBI GI No. 7450927) and Hordeum vulgare (NCBI GI No. 2130042). Shown in Table 6 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”): TABLE 6 BLAST Results for Sequences Encoding Polypeptides Homologous to Magnesium Chelatase Subunit CHLH BLAST Results Clone Status NCBI GI No. pLog Score cdt1c.pk001.o1 FIS 7450927 125.00 chp2.pk0014.h1 EST 2130042 37.40 p0019.clwaa84r EST 2130042 52.30 p0088.clrih26r EST 2130042 40.15 p0110.cgsmo74r EST 2130042 49.70 rlr2.pk0018.e9 FIS 2130042 >254.00 wdk4c.pk005.f24 FIS 2130042 >254.00

[0110] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments, BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a magnesium chelatase subunit CHLH.

Example 5 Characterization of cDNA Clones Encoding Magnesium Chelatase Subunit CHLI

[0111] The BLASTX search using the EST sequences from clones csc1c.pk004.p11, rls48.pk0001.h1 and rlr72.pk0014.f9 revealed similarity of the proteins encoded by the cDNAs to magnesium chelatase subunit CHLI from Glycine max (NCBI GI No. 3334150) with pLog scores of 20.70, 21.52 and 57.00, respectively.

[0112] The sequence of a substantial portion of the cDNA insert from clone csc1c.pk004.p11 is shown in SEQ ID NO:41; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:42. The sequence of a substantial portion of the cDNA insert from clone rls48.pk0001.h1 is shown in SEQ ID NO:47; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:48. The sequence of a substantial portion of the cDNA insert from clone rlr72.pk0014.f9 is shown in SEQ ID NO:45; the deduced amino acid sequence of this substantial portion of the cDNA is shown in SEQ ID NO:46. BLAST scores and probabilities indicate that the instant nucleic acid fragments encode substantial portions of magnesium chelatase subunit CHLI.

[0113] The BLASTX search using the EST sequences from clones listed in Table 7 revealed similarity of the polypeptides encoded by the cDNAs to magnesium chelatase subunit CHLI from Glycine max (NCBI GI No. 3334150). Shown in Table 7 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”): TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous to Magnesium Chelatase Subunit CHLI BLAST pLog Score Clone Status NCBI GI No. 3334150 csc1c.pk004.p11 CGS 164.00 (FIS) rls48.pk0001.h1 CGS 180.00 (FIS)

[0114]FIG. 2 presents an alignment of the amino acid sequences set forth in SEQ ID NOs:44 and 50 and the Glycine max sequence (NCBI GI No. 3334150; SEQ ID NO:52). The data in Table 8 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:44 and 50 and the Glycine max sequence (NCBI GI No. 3334150; SEQ ID NO:52). TABLE 8 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Magnesium Chelatase Subunit CHLI Percent Identity to SEQ ID NO. NCBI GI No. 3334150; SEQ ID NO:52 44 72.6 50 78.1

[0115] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments, BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode all or a substantial portion of a magnesium chelatase subunit CHLI.

Example 6 Expression of Chimeric Genes in Monocot Cells

[0116] A chimeric gene comprising a cDNA encoding the instant polypeptide in sense orientation with respect to the maize 27 kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that is located 3′ to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3′ region.

[0117] The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0118] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0119] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0120] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of mercury (Hg). The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0121] Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0122] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 7 Expression of Chimeric Genes in Dicot Cells

[0123] A seed-specific construct composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin construct includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire construct is flanked by Hind III sites.

[0124] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed construct.

[0125] Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0126] Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0127] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0128] A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed construct comprising the phaseolin 5′ region, the fragment encoding the instant polypeptide and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0129] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0130] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches of mercury (Hg). The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0131] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 8 Expression of Chimeric Genes in Microbial Cells

[0132] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0133] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10 μg/mL ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MASS.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0134] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 9 Evaluating Compounds for Their Ability to Inhibit the Activity of Magnesium Chelatase

[0135] The polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 8, or expression in eukaryotic cell culture, in planta, and using viral expression systems in suitably infected organisms or cell lines. The instant polypeptides may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase (“GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide (“(His)₆”). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzymes. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.

[0136] Purification of the instant polypeptides, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the instant polypeptides are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)₆ peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin.

[0137] Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation of the instant polypeptides disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. For example, assays for magnesium chelatase subunits CHLD, CHLH and CHLI are presented by Papenbrock, J. et al. (1997), Plant J. 12:981-990 and Guo, R. et al. (1998), Plant Physiol. 116:605-615.

1 54 1 432 DNA Zea mays unsure (2) n is a, c, g or t 1 anccaaaaan tgcgtcacna ncctgcgttg gtccngatgc tatcaaaact gctgctgctg 60 cttggggcga ttgatcgtga gatcggaggc attgccatct cagggaagcg tgggacggca 120 aagacagtga tggctcgtgg tttgcatgct atgcttccac ccattgaagt ggtggttggt 180 tccattgcaa atgctgaccc taactcccct gacgaatggg aggatggttt agctgatcaa 240 atacagtatg actctgatgg taatgtcaaa tccgagattg tcaaaacacc ttttgtgcag 300 attccacttg gtgtgacgga ggataggctc attggatcag ttgatgttga agcatctgtg 360 agatcaggga ctactgtatt caacctggct cttgctgaac acataaggtg tcttatgtga 420 tgaataacta tg 432 2 116 PRT Zea mays 2 Lys Leu Leu Leu Leu Leu Gly Ala Ile Asp Arg Glu Ile Gly Gly Ile 1 5 10 15 Ala Ile Ser Gly Lys Arg Gly Thr Ala Lys Thr Val Met Ala Arg Gly 20 25 30 Leu His Ala Met Leu Pro Pro Ile Glu Val Val Val Gly Ser Ile Ala 35 40 45 Asn Ala Asp Pro Asn Ser Pro Asp Glu Trp Glu Asp Gly Leu Ala Asp 50 55 60 Gln Ile Gln Tyr Asp Ser Asp Gly Asn Val Lys Ser Glu Ile Val Lys 65 70 75 80 Thr Pro Phe Val Gln Ile Pro Leu Gly Val Thr Glu Asp Arg Leu Ile 85 90 95 Gly Ser Val Asp Val Glu Ala Ser Val Arg Ser Gly Thr Thr Val Phe 100 105 110 Asn Leu Ala Leu 115 3 2513 DNA Zea mays 3 cggcacgagc catggcgacg cccaccgcgc tccccacctc actcccctac ctcccgcccc 60 gccgcgtcat ctcattccca tccgccgccg ccgtctccct ccccgtcacc tcccgccccg 120 cccggctgcg ggattcccgc ctcgcggccg cggcaacctc ggcctccgag gtcctcgagt 180 ccaccaacgg cgccgtcccc actgcggcca aggacggcgc gtggcgcggg tatgggaggg 240 agtacttccc cctggctgcc gtcgttgggc aggatgctat caaaactgct ctgctgcttg 300 gggcgattga tcgtgagatc ggaggcattg ccatctcagg gaagcgtggg acggcaaaga 360 cagtgatggc tcgtggtttg catgctatgc ttccacccat tgaagtggtg gttggttcca 420 ttgcaaatgc tgaccctaac tcccctgacg aatgggagga tggtttagct gatcaaatac 480 agtatgactc tgatggtaat gtcaaatccg agattgtcaa aacacctttt gtgcagattc 540 cacttggtgt gacggaggat aggctcattg gatcagttga tgttgaagca tctgtgagat 600 cagggactac tgtatttcaa cctggtcttc ttgctgaagc acatagaggt gttctttatg 660 ttgatgaaat aaatctattg gatgatggca taagcaatct acttctgaat gtcttgacgg 720 agggagttaa cattgtggaa agagagggca ttagctttcg ccatccctgc aaaccacttc 780 taattgctac ttacaatcca gaggaagggt ctgtacgtga acacttgctt gatcgtattg 840 caattaattt aagtgctgat cttccaatga gttttgatga ccgcgttgaa gcagtggata 900 ttgcaacacg gtttcaggag tctagcaaag aagttttcaa aatggtggaa gaaaaaactg 960 aaactgcaaa aactcagata atttttgcaa gagagtatct gaaggatgtt actattagca 1020 cagagcagct caaatatctt gtcatggaag ctatacgagg tggctgtcag gggcatcgtg 1080 ctgagttgta tgctgctcga gttgcaaaat gtctagctgc tatggaagga cgtgaaaaag 1140 tatttgtgga tgacctcaag aaagctgtag agctagtcat tctacctcgc tccatcctat 1200 ctgataatcc acaggatcag cagcaagagc aaccaccccc acccccgcca ccaccacctc 1260 cagaaaatca agattcttca gaagaccaag atgaggaaga cgaagaccaa gaggatgatg 1320 aagaagaaaa tgaacaacaa gaccaacaga tacctgagga gttcatattt gatgctgaag 1380 gtggtttagt agatgacaaa cttcttttct ttgcccagca agcacagaga cgacgtggaa 1440 aagctgggcg agcaaagaat gtcatcttct cagaagatag gggccgttac ataaagccta 1500 tgcttcctaa gggtccagta aggaggttag ctgttgatgc cacgcttaga gcagctgcac 1560 cataccaaaa actgcgcaga gagaaagaac gtgacaaaac aagaaaggtt tttgttgaaa 1620 agactgacat gagagccaaa agaatggctc gaaaagcagg tgctctagtc atatttgttg 1680 tggacgctag tggtagcatg gctctgaatc gtatgcagaa tgctaaaggt gcggcgttga 1740 agttgcttgc agaaagctac accagcagag atcaggtttc aattattcct tttcgtggag 1800 attatgctga ggttttgctt ccaccatcaa gatctatagc aatggcccgg aaacgtcttg 1860 agaagctacc atgtggtggt ggttctcctt tagctcatgg cctaagtaca gctgtcagag 1920 tgggtctgaa tgctgaaaag agtggcgatg ttgggcgtat catgattgtt gcaatcaccg 1980 atggaagagc taatgtatca ctgaaggaat caactacccc agaaggtgtt gctgcttcag 2040 atgcgccaag cctttcttct caagaactga aggacgagat acttgaggtg gctggcaaaa 2100 tatacaaggc aggaatgtcc cttcttgtca tcgacactga gaacaagttt gtatccacgg 2160 gatttgccaa ggaaattgca agggttgccc aggggaaata ttattacctc cctaatgctt 2220 cggatgctgt aatttctgct gccaccaaga ccgccctgac agacttgaag agctcatgat 2280 tttgcagcag cggcacccgt tttctgtacc ttttgatagg ggtggtgaac cttcattcat 2340 gcagtagttt ttgtgtaggc ctctacaatg acagggggaa acaaacccga gcatggcatc 2400 gtgtaaagtg ttaaggtcca atggcctcct gtccacgttt ggcgatgtaa atcctccgta 2460 acatagcttg aaccattgag tgtcacgtag tgccatggct agcagttaaa agt 2513 4 755 PRT Zea mays 4 Met Ala Thr Pro Thr Ala Leu Pro Thr Ser Leu Pro Tyr Leu Pro Pro 1 5 10 15 Arg Arg Val Ile Ser Phe Pro Ser Ala Ala Ala Val Ser Leu Pro Val 20 25 30 Thr Ser Arg Pro Ala Arg Leu Arg Asp Ser Arg Leu Ala Ala Ala Ala 35 40 45 Thr Ser Ala Ser Glu Val Leu Glu Ser Thr Asn Gly Ala Val Pro Thr 50 55 60 Ala Ala Lys Asp Gly Ala Trp Arg Gly Tyr Gly Arg Glu Tyr Phe Pro 65 70 75 80 Leu Ala Ala Val Val Gly Gln Asp Ala Ile Lys Thr Ala Leu Leu Leu 85 90 95 Gly Ala Ile Asp Arg Glu Ile Gly Gly Ile Ala Ile Ser Gly Lys Arg 100 105 110 Gly Thr Ala Lys Thr Val Met Ala Arg Gly Leu His Ala Met Leu Pro 115 120 125 Pro Ile Glu Val Val Val Gly Ser Ile Ala Asn Ala Asp Pro Asn Ser 130 135 140 Pro Asp Glu Trp Glu Asp Gly Leu Ala Asp Gln Ile Gln Tyr Asp Ser 145 150 155 160 Asp Gly Asn Val Lys Ser Glu Ile Val Lys Thr Pro Phe Val Gln Ile 165 170 175 Pro Leu Gly Val Thr Glu Asp Arg Leu Ile Gly Ser Val Asp Val Glu 180 185 190 Ala Ser Val Arg Ser Gly Thr Thr Val Phe Gln Pro Gly Leu Leu Ala 195 200 205 Glu Ala His Arg Gly Val Leu Tyr Val Asp Glu Ile Asn Leu Leu Asp 210 215 220 Asp Gly Ile Ser Asn Leu Leu Leu Asn Val Leu Thr Glu Gly Val Asn 225 230 235 240 Ile Val Glu Arg Glu Gly Ile Ser Phe Arg His Pro Cys Lys Pro Leu 245 250 255 Leu Ile Ala Thr Tyr Asn Pro Glu Glu Gly Ser Val Arg Glu His Leu 260 265 270 Leu Asp Arg Ile Ala Ile Asn Leu Ser Ala Asp Leu Pro Met Ser Phe 275 280 285 Asp Asp Arg Val Glu Ala Val Asp Ile Ala Thr Arg Phe Gln Glu Ser 290 295 300 Ser Lys Glu Val Phe Lys Met Val Glu Glu Lys Thr Glu Thr Ala Lys 305 310 315 320 Thr Gln Ile Ile Phe Ala Arg Glu Tyr Leu Lys Asp Val Thr Ile Ser 325 330 335 Thr Glu Gln Leu Lys Tyr Leu Val Met Glu Ala Ile Arg Gly Gly Cys 340 345 350 Gln Gly His Arg Ala Glu Leu Tyr Ala Ala Arg Val Ala Lys Cys Leu 355 360 365 Ala Ala Met Glu Gly Arg Glu Lys Val Phe Val Asp Asp Leu Lys Lys 370 375 380 Ala Val Glu Leu Val Ile Leu Pro Arg Ser Ile Leu Ser Asp Asn Pro 385 390 395 400 Gln Asp Gln Gln Gln Glu Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro 405 410 415 Pro Glu Asn Gln Asp Ser Ser Glu Asp Gln Asp Glu Glu Asp Glu Asp 420 425 430 Gln Glu Asp Asp Glu Glu Glu Asn Glu Gln Gln Asp Gln Gln Ile Pro 435 440 445 Glu Glu Phe Ile Phe Asp Ala Glu Gly Gly Leu Val Asp Asp Lys Leu 450 455 460 Leu Phe Phe Ala Gln Gln Ala Gln Arg Arg Arg Gly Lys Ala Gly Arg 465 470 475 480 Ala Lys Asn Val Ile Phe Ser Glu Asp Arg Gly Arg Tyr Ile Lys Pro 485 490 495 Met Leu Pro Lys Gly Pro Val Arg Arg Leu Ala Val Asp Ala Thr Leu 500 505 510 Arg Ala Ala Ala Pro Tyr Gln Lys Leu Arg Arg Glu Lys Glu Arg Asp 515 520 525 Lys Thr Arg Lys Val Phe Val Glu Lys Thr Asp Met Arg Ala Lys Arg 530 535 540 Met Ala Arg Lys Ala Gly Ala Leu Val Ile Phe Val Val Asp Ala Ser 545 550 555 560 Gly Ser Met Ala Leu Asn Arg Met Gln Asn Ala Lys Gly Ala Ala Leu 565 570 575 Lys Leu Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Ser Ile Ile 580 585 590 Pro Phe Arg Gly Asp Tyr Ala Glu Val Leu Leu Pro Pro Ser Arg Ser 595 600 605 Ile Ala Met Ala Arg Lys Arg Leu Glu Lys Leu Pro Cys Gly Gly Gly 610 615 620 Ser Pro Leu Ala His Gly Leu Ser Thr Ala Val Arg Val Gly Leu Asn 625 630 635 640 Ala Glu Lys Ser Gly Asp Val Gly Arg Ile Met Ile Val Ala Ile Thr 645 650 655 Asp Gly Arg Ala Asn Val Ser Leu Lys Glu Ser Thr Thr Pro Glu Gly 660 665 670 Val Ala Ala Ser Asp Ala Pro Ser Leu Ser Ser Gln Glu Leu Lys Asp 675 680 685 Glu Ile Leu Glu Val Ala Gly Lys Ile Tyr Lys Ala Gly Met Ser Leu 690 695 700 Leu Val Ile Asp Thr Glu Asn Lys Phe Val Ser Thr Gly Phe Ala Lys 705 710 715 720 Glu Ile Ala Arg Val Ala Gln Gly Lys Tyr Tyr Tyr Leu Pro Asn Ala 725 730 735 Ser Asp Ala Val Ile Ser Ala Ala Thr Lys Thr Ala Leu Thr Asp Leu 740 745 750 Lys Ser Ser 755 5 478 DNA Oryza sativa unsure (244) n is a, c, g or t 5 gtgaaaaagt atatgtggat gaccttaaga aagctgtaga gctagttatt ctacctcgat 60 caatcctatc tgataaccca caggagcagc aagaccaaca acctcctcca cccccaccgc 120 caccccctcc acaagatcaa gattctcaag aagatcaaga tgaagacgag gaagaggacc 180 aagaggacga tgatgaagaa aatgaacagc aggaccagca gatacctgag gagttcattt 240 ttgntgctga aggtggtata gtagatgaga agctcctttt ctttgctcag caagctcaaa 300 gacggcgagg gaaagctgga cgagcaaaga atctcatatt ctcatctgat aggggacgat 360 acataggttc tatgcttccc aagggtccaa taaggagggt tagctgttga tgccacactt 420 cgagcagctg caccatacca naaactgagg gngagagaaa agatctgaca agacaagn 478 6 149 PRT Oryza sativa UNSURE (81) Xaa can be any naturally occurring amino acid 6 Glu Lys Val Tyr Val Asp Asp Leu Lys Lys Ala Val Glu Leu Val Ile 1 5 10 15 Leu Pro Arg Ser Ile Leu Ser Asp Asn Pro Gln Glu Gln Gln Asp Gln 20 25 30 Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gln Asp Gln Asp Ser 35 40 45 Gln Glu Asp Gln Asp Glu Asp Glu Glu Glu Asp Gln Glu Asp Asp Asp 50 55 60 Glu Glu Asn Glu Gln Gln Asp Gln Gln Ile Pro Glu Glu Phe Ile Phe 65 70 75 80 Xaa Ala Glu Gly Gly Ile Val Asp Glu Lys Leu Leu Phe Phe Ala Gln 85 90 95 Gln Ala Gln Arg Arg Arg Gly Lys Ala Gly Arg Ala Lys Asn Leu Ile 100 105 110 Phe Ser Ser Asp Arg Gly Arg Tyr Ile Gly Ser Met Leu Pro Lys Gly 115 120 125 Pro Ile Arg Arg Leu Ala Val Asp Ala Thr Leu Arg Ala Ala Ala Pro 130 135 140 Tyr Xaa Lys Leu Arg 145 7 1477 DNA Oryza sativa 7 gcacgaggtg aaaaagtata tgtggatgac cttaagaaag ctgtagagct agttattcta 60 cctcgatcaa tcctatctga taacccacag gagcagcaag accaacaacc tcctccaccc 120 ccaccgccac cccctccaca agatcaagat tctcaagaag atcaagatga agacgaggaa 180 gaggaccaag aggacgatga tgaagaaaat gaacagcagg accagcagat acctgaggag 240 ttcatttttg atgctgaagg tggtatagta gatgagaagc tccttttctt tgctcagcaa 300 gctcaaagac ggcgagggaa agctggacga gcaaagaatc tcatattctc atctgatagg 360 ggacgataca taggttctat gcttcccaag ggtccaataa ggaggttagc tgttgatgcc 420 acacttcgag cagctgcacc ataccagaaa ctgaggagag agaaagatcg tgacaagaca 480 agaaaggttt ttgttgaaaa aactgacatg agagccaaaa gaatggctcg aaaagcaggc 540 gcactggtca tatttgttgt ggatgctagc ggtagcatgg ctctgaatcg catgcagaat 600 gcgaaaggtg cagcattaaa gttgcttgca gaaagctaca caagcagaga tcaggtttca 660 atcattccat ttcgtggaga ttttgctgag gttcttcttc caccttcaag atccatagca 720 atggcccgca atcgtcttga gaagctacca tgtggtggcg gttctccttt agctcacggc 780 cttagcacag ctgtcagagt gggtttgaat gctgaaaaga gcggtgatgt tggacgtatc 840 atgattgttg caatcaccga tggaagagct aatgtgtcac tgaagaaatc gactgaccca 900 gaagccactt cagatgctcc aagaccttct tctcaagaat taaaggatga gatacttgag 960 gtggctggca aaatatacaa ggctggaatt tcacttcttg ttattgatac cgagaacaag 1020 tttgtatcca caggatttgc caaggaaatt gcaagggtcg cccaaggtaa atactattac 1080 ctgccgaatg cttcagacgc tgttatttcc gccgccacca agactgcact ctcggacctg 1140 aagagttcgt gatcctggag agcgttttac cttcagataa tgagtggttt ttacctttta 1200 ccttgtttgg tgcagcagtg tccatgtttc gtgtaacttt gggacgtttc ggctgtgata 1260 accaattttg gcataggatt tttaccgtga gagttggaat tcgggcgtag caccgtgtaa 1320 agaatcatat aatccctctt ctgtctaaat aattggccat gtaaatatgg tgttattgcg 1380 tacagttcta agtaataata acattcataa tttatgtgaa aaaaaaaaaa aaaaaaaaaa 1440 aaaaaaaaaa aaaaaaaaaa aaactcgaga ctagttc 1477 8 383 PRT Oryza sativa 8 Ala Arg Gly Glu Lys Val Tyr Val Asp Asp Leu Lys Lys Ala Val Glu 1 5 10 15 Leu Val Ile Leu Pro Arg Ser Ile Leu Ser Asp Asn Pro Gln Glu Gln 20 25 30 Gln Asp Gln Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gln Asp 35 40 45 Gln Asp Ser Gln Glu Asp Gln Asp Glu Asp Glu Glu Glu Asp Gln Glu 50 55 60 Asp Asp Asp Glu Glu Asn Glu Gln Gln Asp Gln Gln Ile Pro Glu Glu 65 70 75 80 Phe Ile Phe Asp Ala Glu Gly Gly Ile Val Asp Glu Lys Leu Leu Phe 85 90 95 Phe Ala Gln Gln Ala Gln Arg Arg Arg Gly Lys Ala Gly Arg Ala Lys 100 105 110 Asn Leu Ile Phe Ser Ser Asp Arg Gly Arg Tyr Ile Gly Ser Met Leu 115 120 125 Pro Lys Gly Pro Ile Arg Arg Leu Ala Val Asp Ala Thr Leu Arg Ala 130 135 140 Ala Ala Pro Tyr Gln Lys Leu Arg Arg Glu Lys Asp Arg Asp Lys Thr 145 150 155 160 Arg Lys Val Phe Val Glu Lys Thr Asp Met Arg Ala Lys Arg Met Ala 165 170 175 Arg Lys Ala Gly Ala Leu Val Ile Phe Val Val Asp Ala Ser Gly Ser 180 185 190 Met Ala Leu Asn Arg Met Gln Asn Ala Lys Gly Ala Ala Leu Lys Leu 195 200 205 Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Ser Ile Ile Pro Phe 210 215 220 Arg Gly Asp Phe Ala Glu Val Leu Leu Pro Pro Ser Arg Ser Ile Ala 225 230 235 240 Met Ala Arg Asn Arg Leu Glu Lys Leu Pro Cys Gly Gly Gly Ser Pro 245 250 255 Leu Ala His Gly Leu Ser Thr Ala Val Arg Val Gly Leu Asn Ala Glu 260 265 270 Lys Ser Gly Asp Val Gly Arg Ile Met Ile Val Ala Ile Thr Asp Gly 275 280 285 Arg Ala Asn Val Ser Leu Lys Lys Ser Thr Asp Pro Glu Ala Thr Ser 290 295 300 Asp Ala Pro Arg Pro Ser Ser Gln Glu Leu Lys Asp Glu Ile Leu Glu 305 310 315 320 Val Ala Gly Lys Ile Tyr Lys Ala Gly Ile Ser Leu Leu Val Ile Asp 325 330 335 Thr Glu Asn Lys Phe Val Ser Thr Gly Phe Ala Lys Glu Ile Ala Arg 340 345 350 Val Ala Gln Gly Lys Tyr Tyr Tyr Leu Pro Asn Ala Ser Asp Ala Val 355 360 365 Ile Ser Ala Ala Thr Lys Thr Ala Leu Ser Asp Leu Lys Ser Ser 370 375 380 9 398 DNA Glycine max 9 aatgggtttc gctttggcat tcacagcttc ttctacttgc tgctcaaatc tacaatctca 60 gtctctgtta ttcgctgctg ctgcattgag atcaaaaccg tgtctctctc tctgcaacac 120 ttatcgaccc aaacgcattc ggaagcgttc tccaattgtt ggcgctcaat ctgaaaacgg 180 agctctcgtt acttccgaga agcctggcac taattacgga agacaatact tccccctcgc 240 tgctgttgta ggccaagatg ctataaaaac tgctctttta cttggggcca ttgaccctgg 300 gattggagga attgccatat caggaaagcg aggaactgcc aaaactgtaa tggcacgtgg 360 actgcatgca atactgcctc ctattgaagt agtagtag 398 10 131 PRT Glycine max 10 Met Gly Phe Ala Leu Ala Phe Thr Ala Ser Ser Thr Cys Cys Ser Asn 1 5 10 15 Leu Gln Ser Gln Ser Leu Leu Phe Ala Ala Ala Ala Leu Arg Ser Lys 20 25 30 Pro Cys Leu Ser Leu Cys Asn Thr Tyr Arg Pro Lys Arg Ile Arg Lys 35 40 45 Arg Ser Pro Ile Val Gly Ala Gln Ser Glu Asn Gly Ala Leu Val Thr 50 55 60 Ser Glu Lys Pro Gly Thr Asn Tyr Gly Arg Gln Tyr Phe Pro Leu Ala 65 70 75 80 Ala Val Val Gly Gln Asp Ala Ile Lys Thr Ala Leu Leu Leu Gly Ala 85 90 95 Ile Asp Pro Gly Ile Gly Gly Ile Ala Ile Ser Gly Lys Arg Gly Thr 100 105 110 Ala Lys Thr Val Met Ala Arg Gly Leu His Ala Ile Leu Pro Pro Ile 115 120 125 Glu Val Val 130 11 2481 DNA Glycine max 11 gcacgagaat gggtttcgct ttggcattca cagcttcttc tacttgctgc tcaaatctac 60 aatctcagtc tctgttattc gctgctgctg cattgagatc aaaaccgtgt ctctctctct 120 gcaacactta tcgacccaaa cgcattcgga agcgttctcc aattgttggc gctcaatctg 180 aaaacggagc tctcgttact tccgagaagc ctggcactaa ttacggaaga caatacttcc 240 ccctcgctgc tgttgtaggc caagatgcta taaaaactgc tcttttactt ggggccattg 300 accctgggat tggaggaatt gccatatcag gaaagcgagg aactgccaaa actgtaatgg 360 cacgtggact gcatgcaata ctgcctccta ttgaagtagt agtaggttcc atagccaatg 420 cggatccgac ctgcccagaa gagtgggaag atggtcttac agaatgcctg gaatatgatt 480 ctgctggaaa tattaaaact cgtattatca agtctccctt tgttcagatt cctcttggaa 540 tcacggagga cagactcatt ggatcggttg atgttgagga gtctgtgaaa acaggcacaa 600 ctgttttcca gccaggcttg cttgcagaag ctcatagagg tgttttatat gttgatgaaa 660 ttaatctttt ggatgagggt atcagtaatt tgctccttaa tgtattgagt gaaggagtaa 720 atactgttga aagagagggg atcagtttca agcacccttg caggcccctt ctcattgcca 780 cctataaccc agaagagggt gctgttcgtg aacatctgct ggaccgcatt gcgattaatt 840 taagtgcaga tcttccaatg agttttgaaa accgtgttgc agctgttgga attgccacag 900 agtttcagga gaacagtagc caagtatttg agatggtcga agaggaaaca gacaatgcaa 960 aaactcagat catcttggcc agagagtatc taaaagatgt tactctgaac agagatcaat 1020 taaaatacct ggttattgaa gctttacggg gtggttgcca aggacataga gctgagctat 1080 ttgctgcccg tgttgcaaag tgcttagctg ctctggaggg acgggaaaag gtttatgtgg 1140 atgacctaaa aaaagctgta gaattggtca ttctaccccg gtcaatcatt actgagagcc 1200 caccagatca acaaaatcag cctccccccc ctccacctcc tccacaaaat caagaatcag 1260 gcgaagaaca gaatgaagag gaagaacaag aggatgacaa ggatgaagag aatgaacaac 1320 agcaagaaca attacctgaa gagtttatct ttgatgctga aggtggcttg gtagatgaaa 1380 aactcctctt ctttgcccag caagcacaga gacgccgcgg gagggctgga agggcaaaaa 1440 atgtaatatt ttcagaggat agaggccgat acatcaagcc aatgcttcca aagggccctg 1500 taaagagatt agctgtagat gcaaccctta gagctgctgc accttatcaa aaattgcgaa 1560 gggaaaaaga ctcaggaaac agtagaaaag tatttgtgga gaaaacggac atgagggcaa 1620 agagaatggc acgtaaggca ggagcattgg tgatatttgt ggttgatgca agtggaagca 1680 tggcattgaa caggatgcag aatgcaaaag gtgcagcact taagcttctg gctgaaagtt 1740 atacaagcag ggatcaggtc tctataattc cattccgtgg agatgcagct gaagttcttc 1800 tgccaccttc tagatcaatt gcaatggcaa ggaaacgtct tgagaggctt ccatgtggtg 1860 gagggtcccc acttgctcat ggtcttacaa cggctgttag agttggatta aatgcggaga 1920 aaagtggtga cgttggacgt gtgatgattg ttgcaatcac tgatggcaga gccaatatat 1980 cattgaaaag gtcgactgac cctgaagttg ccgcagctac tgatgcccca aaaccttcag 2040 cacaagaatt gaaggatgaa attcttgagg tggctgggaa gatttataaa gcaggaatgt 2100 ctctccttgt catcgacact gaaaataagt ttgtctcaac gggtttcgcc aaggagattg 2160 ctagagttgc ccaaggcaag tattattatc tgccaaatgc ttcagacgct gttatctcat 2220 cggcaacaaa ggaagcttta tcagctttga aaagttcatg aaaccttgta aaaataaagc 2280 agtcaaacat cacttccccc tttggtcgta aatgttaatg taaatcatgc aaactatgtt 2340 atgatgggaa gaacgcatgt tgtaataacc aggagcaatt ttgaatcata ccaatgctaa 2400 caaaccatag acatgataat gatcagtgta aaaaaaaaaa aaaaaaaaaa acaaaaaaaa 2460 aaaaaaaaaa aaaaaaaaaa a 2481 12 750 PRT Glycine max 12 Met Gly Phe Ala Leu Ala Phe Thr Ala Ser Ser Thr Cys Cys Ser Asn 1 5 10 15 Leu Gln Ser Gln Ser Leu Leu Phe Ala Ala Ala Ala Leu Arg Ser Lys 20 25 30 Pro Cys Leu Ser Leu Cys Asn Thr Tyr Arg Pro Lys Arg Ile Arg Lys 35 40 45 Arg Ser Pro Ile Val Gly Ala Gln Ser Glu Asn Gly Ala Leu Val Thr 50 55 60 Ser Glu Lys Pro Gly Thr Asn Tyr Gly Arg Gln Tyr Phe Pro Leu Ala 65 70 75 80 Ala Val Val Gly Gln Asp Ala Ile Lys Thr Ala Leu Leu Leu Gly Ala 85 90 95 Ile Asp Pro Gly Ile Gly Gly Ile Ala Ile Ser Gly Lys Arg Gly Thr 100 105 110 Ala Lys Thr Val Met Ala Arg Gly Leu His Ala Ile Leu Pro Pro Ile 115 120 125 Glu Val Val Val Gly Ser Ile Ala Asn Ala Asp Pro Thr Cys Pro Glu 130 135 140 Glu Trp Glu Asp Gly Leu Thr Glu Cys Leu Glu Tyr Asp Ser Ala Gly 145 150 155 160 Asn Ile Lys Thr Arg Ile Ile Lys Ser Pro Phe Val Gln Ile Pro Leu 165 170 175 Gly Ile Thr Glu Asp Arg Leu Ile Gly Ser Val Asp Val Glu Glu Ser 180 185 190 Val Lys Thr Gly Thr Thr Val Phe Gln Pro Gly Leu Leu Ala Glu Ala 195 200 205 His Arg Gly Val Leu Tyr Val Asp Glu Ile Asn Leu Leu Asp Glu Gly 210 215 220 Ile Ser Asn Leu Leu Leu Asn Val Leu Ser Glu Gly Val Asn Thr Val 225 230 235 240 Glu Arg Glu Gly Ile Ser Phe Lys His Pro Cys Arg Pro Leu Leu Ile 245 250 255 Ala Thr Tyr Asn Pro Glu Glu Gly Ala Val Arg Glu His Leu Leu Asp 260 265 270 Arg Ile Ala Ile Asn Leu Ser Ala Asp Leu Pro Met Ser Phe Glu Asn 275 280 285 Arg Val Ala Ala Val Gly Ile Ala Thr Glu Phe Gln Glu Asn Ser Ser 290 295 300 Gln Val Phe Glu Met Val Glu Glu Glu Thr Asp Asn Ala Lys Thr Gln 305 310 315 320 Ile Ile Leu Ala Arg Glu Tyr Leu Lys Asp Val Thr Leu Asn Arg Asp 325 330 335 Gln Leu Lys Tyr Leu Val Ile Glu Ala Leu Arg Gly Gly Cys Gln Gly 340 345 350 His Arg Ala Glu Leu Phe Ala Ala Arg Val Ala Lys Cys Leu Ala Ala 355 360 365 Leu Glu Gly Arg Glu Lys Val Tyr Val Asp Asp Leu Lys Lys Ala Val 370 375 380 Glu Leu Val Ile Leu Pro Arg Ser Ile Ile Thr Glu Ser Pro Pro Asp 385 390 395 400 Gln Gln Asn Gln Pro Pro Pro Pro Pro Pro Pro Pro Gln Asn Gln Glu 405 410 415 Ser Gly Glu Glu Gln Asn Glu Glu Glu Glu Gln Glu Asp Asp Lys Asp 420 425 430 Glu Glu Asn Glu Gln Gln Gln Glu Gln Leu Pro Glu Glu Phe Ile Phe 435 440 445 Asp Ala Glu Gly Gly Leu Val Asp Glu Lys Leu Leu Phe Phe Ala Gln 450 455 460 Gln Ala Gln Arg Arg Arg Gly Arg Ala Gly Arg Ala Lys Asn Val Ile 465 470 475 480 Phe Ser Glu Asp Arg Gly Arg Tyr Ile Lys Pro Met Leu Pro Lys Gly 485 490 495 Pro Val Lys Arg Leu Ala Val Asp Ala Thr Leu Arg Ala Ala Ala Pro 500 505 510 Tyr Gln Lys Leu Arg Arg Glu Lys Asp Ser Gly Asn Ser Arg Lys Val 515 520 525 Phe Val Glu Lys Thr Asp Met Arg Ala Lys Arg Met Ala Arg Lys Ala 530 535 540 Gly Ala Leu Val Ile Phe Val Val Asp Ala Ser Gly Ser Met Ala Leu 545 550 555 560 Asn Arg Met Gln Asn Ala Lys Gly Ala Ala Leu Lys Leu Leu Ala Glu 565 570 575 Ser Tyr Thr Ser Arg Asp Gln Val Ser Ile Ile Pro Phe Arg Gly Asp 580 585 590 Ala Ala Glu Val Leu Leu Pro Pro Ser Arg Ser Ile Ala Met Ala Arg 595 600 605 Lys Arg Leu Glu Arg Leu Pro Cys Gly Gly Gly Ser Pro Leu Ala His 610 615 620 Gly Leu Thr Thr Ala Val Arg Val Gly Leu Asn Ala Glu Lys Ser Gly 625 630 635 640 Asp Val Gly Arg Val Met Ile Val Ala Ile Thr Asp Gly Arg Ala Asn 645 650 655 Ile Ser Leu Lys Arg Ser Thr Asp Pro Glu Val Ala Ala Ala Thr Asp 660 665 670 Ala Pro Lys Pro Ser Ala Gln Glu Leu Lys Asp Glu Ile Leu Glu Val 675 680 685 Ala Gly Lys Ile Tyr Lys Ala Gly Met Ser Leu Leu Val Ile Asp Thr 690 695 700 Glu Asn Lys Phe Val Ser Thr Gly Phe Ala Lys Glu Ile Ala Arg Val 705 710 715 720 Ala Gln Gly Lys Tyr Tyr Tyr Leu Pro Asn Ala Ser Asp Ala Val Ile 725 730 735 Ser Ser Ala Thr Lys Glu Ala Leu Ser Ala Leu Lys Ser Ser 740 745 750 13 511 DNA Glycine max unsure (32) n is a, c, g or t 13 ttgggattga gtgaagaaac agtggtcgtt cnctctgaaa tgggtttcgc tttggcatac 60 acagcntntg ggttgtngct caaacctaca atttcagtct ctgttattcg ctgctgcttc 120 attgagatca aaaccgtgtc tctctctctg caactctact tatcgaccca aacgcattct 180 ccagcgttct ccaattgttg gcgctcagtc tgaaaatgga gctctggtta cttcggagaa 240 gcccgacact aattacggaa gacaatactt ccccctcgct gctgttgtag gccaagattc 300 tataaaaact gctcttttac ttggtgcaat tgaccccggg gntggaggaa ttgccatatc 360 aggaaagcga ggaatgccan aactgtaatg gcacgtggat tgatgaatac tgctcctatt 420 gagggagagg ttcattgcaa tnggatcaac tgccagaang gggaantgtc tacagatgct 480 gatatntctc nggaattann ctcgatacna n 511 14 109 PRT Glycine max UNSURE (7)..(8) Xaa can be any naturally occurring amino acid 14 Ser Leu Trp His Thr Gln Xaa Xaa Gly Cys Xaa Ser Asn Leu Gln Phe 1 5 10 15 Gln Ser Leu Leu Phe Ala Ala Ala Ser Leu Arg Ser Lys Pro Cys Leu 20 25 30 Ser Leu Cys Asn Ser Thr Tyr Arg Pro Lys Arg Ile Leu Gln Arg Ser 35 40 45 Pro Ile Val Gly Ala Gln Ser Glu Asn Gly Ala Leu Val Thr Ser Glu 50 55 60 Lys Pro Asp Thr Asn Tyr Gly Arg Gln Tyr Phe Pro Leu Ala Ala Val 65 70 75 80 Val Gly Gln Asp Ser Ile Lys Thr Ala Leu Leu Leu Gly Ala Ile Asp 85 90 95 Pro Gly Xaa Gly Gly Ile Ala Ile Ser Gly Lys Arg Gly 100 105 15 438 DNA Triticum aestivum unsure (401) n is a, c, g or t 15 ggcagcatgg ctctgaaccg catgcagaat gccaaaggtg cagcattgaa gttgcttgca 60 gaaagttaca caagcagaga tcaggttgca attattccct tccgtggaga ctatgctgag 120 gttctgcttc caccatcaag atccattgca atggctcgca aacgtcttga aaagctacca 180 tgcggtggcg gttctccttt agctcatggc ctgagtacag ctgtcagagt gggattgaac 240 gctgaaaaga gtggcgacgt tgggcgtatc atggaattcg gttggccaaa tcacccggat 300 ggaaagaagc taaatggtta tcaactggaa gaaatccaaa tgacccagaa gctgcagctg 360 cttcagacgc accaagacca tctactcaag aattgaaggg ngagatactt gatgtgtctg 420 cagggagatc agggcagg 438 16 91 PRT Triticum aestivum 16 Gly Ser Met Ala Leu Asn Arg Met Gln Asn Ala Lys Gly Ala Ala Leu 1 5 10 15 Lys Leu Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Ala Ile Ile 20 25 30 Pro Phe Arg Gly Asp Tyr Ala Glu Val Leu Leu Pro Pro Ser Arg Ser 35 40 45 Ile Ala Met Ala Arg Lys Arg Leu Glu Lys Leu Pro Cys Gly Gly Gly 50 55 60 Ser Pro Leu Ala His Gly Leu Ser Thr Ala Val Arg Val Gly Leu Asn 65 70 75 80 Ala Glu Lys Ser Gly Asp Val Gly Arg Ile Met 85 90 17 912 DNA Triticum aestivum 17 gcacgagggc agcatggctc tgaaccgcat gcagaatgcc aaaggtgcag cattgaagtt 60 gcttgcagaa agttacacaa gcagagatca ggttgcaatt attcccttcc gtggagacta 120 tgctgaggtt ctgcttccac catcaagatc cattgcaatg gctcgcaaac gtcttgaaaa 180 gctaccatgc ggtggcggtt ctcctttagc tcatggcctg agtacagctg tcagagtggg 240 attgaacgct gaaaagagtg gcgacgttgg gcgtatcatg atcgttgcaa tcaccgatgg 300 aagagctaat gtatcactga agaaatccaa tgacccagaa gctgcagctg cttcagacgc 360 accaagacca tctactcaag aattgaagga tgagatactt gatgtgtctg caaaaatatt 420 caaagcagga atgtcgcttc tcgtcatcga taccgagaac aagtttgtat ctacgggatt 480 cgccaaggaa atcgcaaggg ttgcccaagg gaaatactac tacctgccaa acgcttcgga 540 cgccgtgatt tcggccgcca ccaagaccgc gctggcggac ttgaagagct agagagcgat 600 ctcggagcgt cgatcagcac ccgcccaact attgtttgta ccgtctgatg ataaaagttg 660 tttcgtgcag taatttgtgc agctgtcctt agttctttct gtaacttttt tgggacgtgc 720 gtttcagctc ttatgaccca attttggtgt aggttttctt ttcttctttc tttctctttt 780 accagcacca gcagggctaa agtccgagca taacttcgtg taaatgtcgg aattctccca 840 ctgcctctct gataattggc cttgtaaatc tactgctgtt aattttcgag gcagaaaaaa 900 aaaaaaaaaa aa 912 18 196 PRT Triticum aestivum 18 His Glu Gly Ser Met Ala Leu Asn Arg Met Gln Asn Ala Lys Gly Ala 1 5 10 15 Ala Leu Lys Leu Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Ala 20 25 30 Ile Ile Pro Phe Arg Gly Asp Tyr Ala Glu Val Leu Leu Pro Pro Ser 35 40 45 Arg Ser Ile Ala Met Ala Arg Lys Arg Leu Glu Lys Leu Pro Cys Gly 50 55 60 Gly Gly Ser Pro Leu Ala His Gly Leu Ser Thr Ala Val Arg Val Gly 65 70 75 80 Leu Asn Ala Glu Lys Ser Gly Asp Val Gly Arg Ile Met Ile Val Ala 85 90 95 Ile Thr Asp Gly Arg Ala Asn Val Ser Leu Lys Lys Ser Asn Asp Pro 100 105 110 Glu Ala Ala Ala Ala Ser Asp Ala Pro Arg Pro Ser Thr Gln Glu Leu 115 120 125 Lys Asp Glu Ile Leu Asp Val Ser Ala Lys Ile Phe Lys Ala Gly Met 130 135 140 Ser Leu Leu Val Ile Asp Thr Glu Asn Lys Phe Val Ser Thr Gly Phe 145 150 155 160 Ala Lys Glu Ile Ala Arg Val Ala Gln Gly Lys Tyr Tyr Tyr Leu Pro 165 170 175 Asn Ala Ser Asp Ala Val Ile Ser Ala Ala Thr Lys Thr Ala Leu Ala 180 185 190 Asp Leu Lys Ser 195 19 583 DNA Zea mays unsure (397) n is a, c, g or t 19 gtcgtccgta accgccgccg tgcagcagct caacgccgac ccgcgccgcg ccgccgcgtt 60 cgaggtcgtg ggctacctcg tcgaggagct ccgcgacgag gacacctacg ccaccttctg 120 cgccgacctc gccgacgcca acgtcttcat cggctccctc atcttcgtcg aggagctggc 180 cctcaaggtc aaggccgccg tcgagaagga gcgcgaccgc atggacgccg tcctcgtctt 240 cccctcaatg cccgaggtca tgcgcctcaa caagctcggc tccttcagca tgtcgcaagc 300 tggggcagtc caaagagccc cttcttccag ctcttcaagc gcaacaaggg caactccaag 360 caactttcgc cgacaagaat gctcaaagct cgtccgnaag gctgccaaag gtgctcaaag 420 taacttggcc tctgacaaag gngcaaggac gccccggggt ctanattctn aagcttcaan 480 tctgggtccg tgggtccccc ggncaaactc caannatttc tcnaagatga ttgccgggtn 540 ctaaatggct ggcctcaaag ggggccggat caantacgaa gan 583 20 118 PRT Zea mays 20 Ser Ser Val Thr Ala Ala Val Gln Gln Leu Asn Ala Asp Arg Arg Ala 1 5 10 15 Ala Ala Phe Glu Val Val Gly Tyr Leu Val Glu Glu Leu Arg Asp Glu 20 25 30 Asp Thr Tyr Ala Thr Phe Cys Ala Asp Leu Ala Asp Ala Asn Val Phe 35 40 45 Ile Gly Ser Leu Ile Phe Val Glu Glu Leu Ala Leu Lys Val Lys Ala 50 55 60 Ala Val Glu Lys Glu Arg Asp Arg Met Asp Ala Val Leu Val Phe Pro 65 70 75 80 Ser Met Pro Glu Val Met Arg Leu Asn Lys Leu Gly Ser Phe Ser Met 85 90 95 Ser Gln Ala Gly Gly Ser Pro Lys Ser Pro Phe Phe Gln Leu Phe Lys 100 105 110 Arg Asn Lys Gly Asn Ser 115 21 782 DNA Zea mays 21 gcacgaggtc gtccgtaacc gccgccgtgc agcagctcaa cgccgacccg cgccgcgccg 60 ccgcgttcga ggtcgtgggc tacctcgtcg aggagctccg cgacgaggac acctacgcca 120 ccttctgcgc cgacctcgcc gacgccaacg tcttcatcgg ctccctcatc ttcgtcgagg 180 agctggccct caaggtcaag gccgccgtcg agaaggagcg cgaccgcatg gacgccgtcc 240 tcgtcttccc ctcaatgccc gaggtcatgc gcctcaacaa gctcggctcc ttcagcatgt 300 cgcagctggg gcagtccaag agccccttct tccagctctt caagcgcaac aaggccaact 360 ccagcaactt cgccgacagc atgctcaagc tcgtccgcac gctgcccaag gtgctcaagt 420 acctgccctc tgacaaggcg caggacgccc ggctctacat cctcagcctc cagttctggc 480 tcggtggctc gccggacaac ctccagaact tcctcaagat gatcgccggc tcctacgtgc 540 ctgccctcaa gggcgccggc atcaagtacg acgaccccgt tctctacctc gactccggca 600 tctggcaccc gctggcgccc accatgtacg aggacgtcaa ggagtacctc aactggtacg 660 gcacgcgccg ggacgccaac gacaggctca aggaccccaa ggcgcccatc atcggcctcg 720 tcctgcagag gagccacatt gtcaccggcg acgacgggca ctacgtcgcc gtcatcatgg 780 ag 782 22 260 PRT Zea mays 22 Thr Arg Ser Ser Val Thr Ala Ala Val Gln Gln Leu Asn Ala Asp Pro 1 5 10 15 Arg Arg Ala Ala Ala Phe Glu Val Val Gly Tyr Leu Val Glu Glu Leu 20 25 30 Arg Asp Glu Asp Thr Tyr Ala Thr Phe Cys Ala Asp Leu Ala Asp Ala 35 40 45 Asn Val Phe Ile Gly Ser Leu Ile Phe Val Glu Glu Leu Ala Leu Lys 50 55 60 Val Lys Ala Ala Val Glu Lys Glu Arg Asp Arg Met Asp Ala Val Leu 65 70 75 80 Val Phe Pro Ser Met Pro Glu Val Met Arg Leu Asn Lys Leu Gly Ser 85 90 95 Phe Ser Met Ser Gln Leu Gly Gln Ser Lys Ser Pro Phe Phe Gln Leu 100 105 110 Phe Lys Arg Asn Lys Ala Asn Ser Ser Asn Phe Ala Asp Ser Met Leu 115 120 125 Lys Leu Val Arg Thr Leu Pro Lys Val Leu Lys Tyr Leu Pro Ser Asp 130 135 140 Lys Ala Gln Asp Ala Arg Leu Tyr Ile Leu Ser Leu Gln Phe Trp Leu 145 150 155 160 Gly Gly Ser Pro Asp Asn Leu Gln Asn Phe Leu Lys Met Ile Ala Gly 165 170 175 Ser Tyr Val Pro Ala Leu Lys Gly Ala Gly Ile Lys Tyr Asp Asp Pro 180 185 190 Val Leu Tyr Leu Asp Ser Gly Ile Trp His Pro Leu Ala Pro Thr Met 195 200 205 Tyr Glu Asp Val Lys Glu Tyr Leu Asn Trp Tyr Gly Thr Arg Arg Asp 210 215 220 Ala Asn Asp Arg Leu Lys Asp Pro Lys Ala Pro Ile Ile Gly Leu Val 225 230 235 240 Leu Gln Arg Ser His Ile Val Thr Gly Asp Asp Gly His Tyr Val Ala 245 250 255 Val Ile Met Glu 260 23 507 DNA Zea mays unsure (79) n is a, c, g or t 23 tattgcccac gaccaagttc gtcagagcgg acagagagaa gatgagggtt ctgtttgggt 60 tcttggggga gtgcctgang ctcgtcgtgc aagacaacga gctgggaagc ttgaagcttg 120 ccctcgaggg aagctacgtc gagcctggac ctggcggcga cccgatccgt aacccgaagt 180 gctcccgaca ggaagaacat ccacgctctc gatccgcagg ccatcccaac cacggctgcc 240 ttgaagagcg ccaagatcgt cgtggaccgt ctcctggaga ggcagaaggc tgacaatggc 300 ggcaagtacc ctgagacggt cgcacttgtc ctgtggggca ccgacaacat caagacctat 360 ggtgagtcac tagcccaggt gctgtggatg attggagttc ggccagttgc cgacaccttc 420 ggccgtgtca accgtgtgga gcctgtcagc cttgaggagc ttggacgccc aaggatcgat 480 gtcgtcgtca attgctcngg gtgtttt 507 24 108 PRT Zea mays 24 Pro Asp Arg Lys Asn Ile His Ala Leu Asp Pro Gln Ala Ile Pro Thr 1 5 10 15 Thr Ala Ala Leu Lys Ser Ala Lys Ile Val Val Asp Arg Leu Leu Glu 20 25 30 Arg Gln Lys Ala Asp Asn Gly Gly Lys Tyr Pro Glu Thr Val Ala Leu 35 40 45 Val Leu Trp Gly Thr Asp Asn Ile Lys Thr Tyr Gly Glu Ser Leu Ala 50 55 60 Gln Val Leu Trp Met Ile Gly Val Arg Pro Val Ala Asp Thr Phe Gly 65 70 75 80 Arg Val Asn Arg Val Glu Pro Val Ser Leu Glu Glu Leu Gly Arg Pro 85 90 95 Arg Ile Asp Val Val Val Asn Cys Ser Gly Cys Phe 100 105 25 351 DNA Zea mays unsure (202) n is a, c, g or t 25 tattgcacac agcgctacct ggtcgacccg attaccggca agacgttcgt gaacgccgtg 60 gtgtctctca ccgggttcgc gctcgtcggg gggccggcga ggcaggacca tcccaaggcc 120 attgccgcgc tgcagaagct cgacgtgccg tacattgtcg cgctcccgct cgtgttccag 180 accacggagg agtggctcaa cnagcacctt ggggcttcac ccaattcagg tggcgctgca 240 ggtcgcgctg ccggagntcg acggtgngat ggagcccatt cgtngttncg ccggcggana 300 nccccaagga caggggaagt cccaatgcat tgnacaagag acttggagca g 351 26 103 PRT Zea mays UNSURE (87) Xaa can be any naturally occurring amino acid 26 Gln Arg Tyr Leu Val Asp Pro Ile Thr Gly Lys Thr Phe Val Asn Ala 1 5 10 15 Val Val Ser Leu Thr Gly Phe Ala Leu Val Gly Gly Pro Ala Arg Gln 20 25 30 Asp His Pro Lys Ala Ile Ala Ala Leu Gln Lys Leu Asp Val Pro Tyr 35 40 45 Ile Val Ala Leu Pro Leu Val Phe Gln Thr Thr Glu Glu Trp Leu Asn 50 55 60 Glu Trp Leu Asn Ser Thr Leu Gly Leu His Pro Ile Gln Val Ala Leu 65 70 75 80 Gln Val Ala Leu Pro Glu Xaa Asp Gly Xaa Met Glu Pro Ile Arg Xaa 85 90 95 Xaa Ala Gly Gly Xaa Pro Gln 100 27 439 DNA Zea mays 27 ggcacgccca ggagcaggcg gaggagctcg gcgtgtcgct aagggaggcg gcgacaaggg 60 tgttctcgaa cgcatcaggc tcctactcgt ccaacgtgaa cctggcggtg gagaacgcgt 120 catggaccga cgagaagcag ctccaggaca tgtacctgag ccgcaagtcc ttcgcgttcg 180 acagcgacgc ccctggggca ggcatgaagg agaagcgcaa ggcgttcgag ctcgccctgg 240 cgacggcgga cgccacgttc cagaacctcg actcgtcgga gatctcgctg acggacgtga 300 gccactactt tcgacttcgg acccgaccaa gctcgtgcag gggcttgcgc aaggacgggc 360 gggcgccgtc cttcgtacat aagccgacac caacacggcg aacgcccaag tgaagacgct 420 gtcggagaag gtgcgcctt 439 28 136 PRT Zea mays 28 His Ala Gln Glu Gln Ala Glu Glu Leu Gly Val Ser Leu Arg Glu Ala 1 5 10 15 Ala Thr Arg Val Phe Ser Asn Ala Ser Gly Ser Tyr Ser Ser Asn Val 20 25 30 Asn Leu Ala Val Glu Asn Ala Ser Trp Thr Asp Glu Lys Gln Leu Gln 35 40 45 Asp Met Tyr Leu Ser Arg Lys Ser Phe Ala Phe Asp Ser Asp Ala Pro 50 55 60 Gly Ala Gly Met Lys Glu Lys Arg Lys Ala Phe Glu Leu Ala Leu Ala 65 70 75 80 Thr Ala Asp Ala Thr Phe Gln Asn Leu Asp Ser Ser Glu Ile Ser Leu 85 90 95 Thr Asp Val Ser His Tyr Phe Arg Leu Arg Thr Arg Pro Ser Ser Cys 100 105 110 Arg Gly Leu Arg Lys Asp Gly Arg Ala Pro Ser Phe Val His Lys Pro 115 120 125 Thr Pro Thr Arg Arg Thr Pro Lys 130 135 29 378 DNA Zea mays unsure (27) n is a, c, g or t 29 gaccgccccg aggacggcat aacctcnctg cccggcatac ttgccgccac agtgggcagg 60 gacattgaan atgtgtacag gggaagtgac aagggcatac tggctgacnt cgancttctg 120 aggcagatca ctgaggcttc gcgcggcgcc atcaccgcct tcnttgagaa gaccacaaac 180 agcaaagggc aggtcgtcaa tgttaccaac aacctcagca agatacttgg tttcggtctg 240 tcggaaccat gggtgcaata nctgtncacg accaagttcg tcagagcggg anagagagaa 300 gatgnagggt tctgtttggg ttcttagggg agtgcctgaa gntcgtcgtg ccaaanaacg 360 agctggggaa agcttgaa 378 30 108 PRT Zea mays UNSURE (24) Xaa can be any naturally occurring amino acid 30 Asp Arg Pro Glu Asp Gly Ile Thr Ser Leu Pro Gly Ile Leu Ala Ala 1 5 10 15 Thr Val Gly Arg Asp Ile Glu Xaa Val Tyr Arg Gly Ser Asp Lys Gly 20 25 30 Ile Leu Ala Asp Xaa Xaa Leu Leu Arg Gln Ile Thr Glu Ala Ser Arg 35 40 45 Gly Ala Ile Thr Ala Phe Xaa Glu Lys Thr Thr Asn Ser Lys Gly Gln 50 55 60 Val Val Asn Val Thr Asn Asn Leu Ser Lys Ile Leu Gly Phe Gly Leu 65 70 75 80 Ser Glu Pro Trp Val Gln Xaa Leu Xaa Thr Thr Lys Phe Val Arg Ala 85 90 95 Gly Xaa Arg Glu Asp Xaa Gly Phe Cys Leu Gly Ser 100 105 31 495 DNA Zea mays unsure (9) n is a, c, g or t 31 ggtacgaang tgatcccatg cggcttctct tctcaaagtc tgccagccct caccatggat 60 ttgcagcata ctacaccttt gtcgagaaga tcttccaggc cgatgctgtt ctgcactttg 120 gaacacacgg gtccctcgag ttcatgcctg gcaagcaggt tgggatgagt gacgcctgct 180 tccctgacag cctcattggc aacatcccca acatctacta ctatgctgca aacaacccat 240 cagaagccac ggtgggccaa gcgccgganc tacgcgaaca ccatcagcta cctgaaccca 300 ccgggcgaaa aacgccgggc tctacaaggg gctcaagcag ctgttcagaa ctcatctctt 360 cctaccantc tcttcaagga naccggggtt gtcctcaaat tgtnaactcc atcgtcagca 420 ctgcaaacaa tgcaacctcc aaaagatttt ncgctgnccn aagaanggga agaatcccac 480 caaagaactt aactt 495 32 164 PRT Zea mays UNSURE (3) Xaa can be any naturally occurring amino acid 32 Tyr Glu Xaa Asp Pro Met Arg Leu Leu Phe Ser Lys Ser Ala Ser Pro 1 5 10 15 His His Gly Phe Ala Ala Tyr Tyr Thr Phe Val Glu Lys Ile Phe Gln 20 25 30 Ala Asp Ala Val Leu His Phe Gly Thr His Gly Ser Leu Glu Phe Met 35 40 45 Pro Gly Lys Gln Val Gly Met Ser Asp Ala Cys Phe Pro Asp Ser Leu 50 55 60 Ile Gly Asn Ile Pro Asn Ile Tyr Tyr Tyr Ala Ala Asn Asn Pro Ser 65 70 75 80 Glu Ala Thr Val Gly Gln Ala Pro Xaa Leu Arg Glu His His Gln Leu 85 90 95 Pro Glu Pro Thr Gly Arg Lys Thr Pro Gly Ser Thr Arg Gly Ser Ser 100 105 110 Ser Cys Ser Glu Leu Ile Ser Ser Tyr Xaa Ser Leu Gln Gly Xaa Arg 115 120 125 Gly Cys Pro Gln Ile Val Asn Ser Ile Val Ser Thr Ala Asn Asn Ala 130 135 140 Thr Ser Lys Arg Phe Xaa Ala Xaa Xaa Arg Xaa Gly Arg Ile Pro Pro 145 150 155 160 Lys Asn Leu Thr 33 652 DNA Oryza sativa unsure (347) n is a, c, g or t 33 atcactcctc aagggcgccg acatcaagta cgacgacccc gtcctcttcc tcgacgctgg 60 tatctggcac ccgctggcgc ccaccatgta cgacgacgtc aaggagtacc tcaactggta 120 cggcacccgc cgcgacacca acgacaagct caaggacccc aacgcgccgg tgatcggcct 180 cgttttgcag aggagccaca ttgtcaccgg agacgacggt cactacgtcg ccgtgatcat 240 ggagctggag gccaagggtg ccaaggtcat accgatcttc gccggcgggc tgggacttct 300 cggggaccca cgcagcgggt acctcgtcaa cccgatcacc gggaaanctt cgtgaacgcg 360 gtgggtgtcg ctcaccgggt tcgcgctcgt cngagggcan cgagcaagac atcccaagng 420 nngccgcgct gcaaaactcg actgccgtca tcgtggatgc cctcgtgtca aacnaagaag 480 atggtgaaca caatgggctg ancnaataag tgngctcaag tgcctcccgg actnacgtgg 540 aaggaccatg gttcccggcg taccaaaaag gaataaatct tgaaaaggng gcacntncaa 600 acatnantgg aaactaaagg aanaggggaa aacgaacncg tttnctcnaa aa 652 34 114 PRT Oryza sativa UNSURE (113) Xaa can be any naturally occurring amino acid 34 Leu Lys Gly Ala Asp Ile Lys Tyr Asp Asp Pro Val Leu Phe Leu Asp 1 5 10 15 Ala Gly Ile Trp His Pro Leu Ala Pro Thr Met Tyr Asp Asp Val Lys 20 25 30 Glu Tyr Leu Asn Trp Tyr Gly Thr Arg Arg Asp Thr Asn Asp Lys Leu 35 40 45 Lys Asp Pro Asn Ala Pro Val Ile Gly Leu Val Leu Gln Arg Ser His 50 55 60 Ile Val Thr Gly Asp Asp Gly His Tyr Val Ala Val Ile Met Glu Leu 65 70 75 80 Glu Ala Lys Gly Ala Lys Val Ile Pro Ile Phe Ala Gly Gly Leu Gly 85 90 95 Leu Leu Gly Pro Arg Ser Gly Tyr Leu Val Asn Pro Ile Thr Gly Lys 100 105 110 Xaa Ser 35 3521 DNA Oryza sativa 35 gcacgagatc actcctcaag ggcgccgaca tcaagtacga cgaccccgtc ctcttcctcg 60 acgctggtat ctggcacccg ctggcgccca ccatgtacga cgacgtcaag gagtacctca 120 actggtacgg cacccgccgc gacaccaacg acaagctcaa ggaccccaac gcgccggtga 180 tcggcctcgt tttgcagagg agccacattg tcaccggaga cgacggtcac tacgtcgccg 240 tgatcatgga gctggaggcc aagggtgcca aggtcatacc gatcttcgcc ggcgggctgg 300 acttctcggg acccacgcag cggtacctcg tcgacccgat caccggaaag ccgttcgtga 360 acgcggtggt gtcgctcacc gggttcgcgc tcgtcggagg gccagcgagg caggaccatc 420 ccaaggcgat cgccgcgctg cagaagctcg acgtgccgta catcgtggca ctgccgctcg 480 tgttccagac gacagaggag tggctgaaca gcacattggg cctgcacccg attcaggtgg 540 cgctgcaggt tgcgctcccg gagcttgacg gtggcatgga gcccattgtg ttcgccggcc 600 gtgaccccag aacagggaag tcacatgcgt tgcacaagag ggtggagcag ctctgcacta 660 gagcaatcag atgggcagag ctgaagagga aaactaagga ggagaagaaa ctggcaatca 720 ctgttttcag cttcccacca gacaaaggca atgttggcac agcagcatac ctgaatgttt 780 tcaactccat ctactccgtc ctccaagatc tgaagaagga tggctacaat gttgagggtc 840 ttccagacac agctgaggcc ctcatcgagg aggttattca tgataaggag gcccaattca 900 atagccccaa cctcaatgtt gcttaccgca tgaacgtgcg ggagtaccag tcactcactt 960 cctatgcctc cttgctggag gagaactggg gcaagccacc tgggaacctt aattctgatg 1020 gtgaaaacct ccttgtctat gggaaacagt acggcaatgt attcattgga gttcagccca 1080 cttttggcta tgaaggagat ccgatgcggc ttctgttctc aaaatctgct agccctcacc 1140 atggctttgc agcatactac acctttgttg agaagatctt ccaggctgat gctgttcttc 1200 actttggtac ccatgggtct cttgagttca tgccagggaa gcaggttggg atgagtgatg 1260 catgctatcc tgacagtctc attggcaaca tccccaatat ctactactat gcagcaaaca 1320 atccatcaga agcaactgtt gccaagcgca gaagctatgc aaacaccata agctacctga 1380 caccaccagc tgaaaatgct ggtctctaca aggggctcaa gcagctttca ggagctcatc 1440 tcttcttaac caatctctca aggacacagg acgtggtccg cagattgtga gctcaatcat 1500 tagcactgca aaacagtgta atcttgacaa ggatgttccc ttgcctgagg aaggtgtgga 1560 gcttccacca aatgagcgtg accttattgt tggaaaggtg tatgccaaga tcatggaaat 1620 agaatcacgc ctcctaccat gcggtctgca tgtgataggt gagccaccaa gtgccatcga 1680 ggctgtggcc accttggtga acatagcttc ccttgatcgc ccagaggatg aaatatactc 1740 actgcctaac atacttgctc agacagtggg caggaacatt gaagatgtgt acagaggaag 1800 tgacaaggga atactggcgg atgttgaact gttgaggcag ataacagaag cttcacgtgg 1860 tgccatcact acctttgttg agaggactac aaacaacaaa gggcaagttg ttgatgttac 1920 aaacaaactt agtaccatgc ttggttttgg tttatcagaa ccatgggtac aacacttgtc 1980 caagaccaag ttcatcagag cagacagaga gaaattgaga accttgttta ctttcttggg 2040 agaatgcttg aagctaattg tggcagataa tgagctggga agcttgaaac ttgccctcga 2100 gggaagctat gttgaacctg gccctggtgg tgatccaatc cgtaacccga aggttctccc 2160 gacagggaag aacatccatg ctcttgaccc tcaggcaatc ccaactacag ctgccttgaa 2220 gagcgccaaa attattgtag accgtctgct ggagcggcaa aaggttgaca atggtggcaa 2280 gtatcctgag acaattgcac ttgtcttgtg gggcaccgat aacatcaaga cctatggtga 2340 gtcattggcc caggtgctgt ggatgattgg tgtgcgcccg gttgctgaca cctttggccg 2400 tgtcaaccgt gtggaacctg tcagccttga ggagcttgga cgtcccagga ttgacgttgt 2460 tatcaactgc tcgggtgtct tcagagatct tttcatcaac cagatgaatc tactggaccg 2520 ggcagtgaag atggttgccg aactggatga gccagaagag atgaactacg tgcgtaagca 2580 tgcacaagag caggcacggg aacttggcgt ttcattaaga gaggcggcaa caagggtgtt 2640 ctcaaatgca tcaggctctt actcatcgaa tgtgaacttg gcagtggaga atgcatcatg 2700 gactgatgag aagcagctcc aggacatgta cctgagtcgc aagtcttttg catttgattg 2760 tgatgctcca ggggcaggca tgcgagagca acgcaagaca tttgagcttg ctctagcaac 2820 agcagatgcc acattccaga acctagactc atcagagatt tcactaacag atgtgagcca 2880 ctactttgac tcagacccga caaagctggt gcaaggactg cgcaaggatg ggcgggcacc 2940 ttcctcatac atagcagata caaccacagc aaatgcacag gtgaggacat tgtcagagac 3000 agtgcgcctt gatgcaagga caaagctact gaaccctaag tggtacgagg ggatgatgaa 3060 aagtggctac gagggagtta gagagattga gaagcggctg acaaatactg ttggatggag 3120 tgcaacatct ggacaggttg acaactgggt ttatgaggag gcaaatgcca catttattga 3180 agatgaggct atgaggaaga ggctcatgga caccaacccc aattcattca ggaagctagt 3240 tcagaccttc ctagaagcca gtggcagagg ctactgggag acatcagagg aaaacttgga 3300 aaagctcagg gagctctact ctgaggttga agacaagatt gaaggaattg accggtaaat 3360 ttatttgatc tatcagatcc tgcattcaac caaggaggag aaatccttct gtctcactga 3420 atctagagtt gagacttgta cactttgtat aatttataaa aagttgtaac atgacataca 3480 cgaggatacc gtgttttaac aaaaaaaaaa aaaaaaaaaa a 3521 36 1118 PRT Oryza sativa 36 Thr Arg Ser Leu Leu Lys Gly Ala Asp Ile Lys Tyr Asp Asp Pro Val 1 5 10 15 Leu Phe Leu Asp Ala Gly Ile Trp His Pro Leu Ala Pro Thr Met Tyr 20 25 30 Asp Asp Val Lys Glu Tyr Leu Asn Trp Tyr Gly Thr Arg Arg Asp Thr 35 40 45 Asn Asp Lys Leu Lys Asp Pro Asn Ala Pro Val Ile Gly Leu Val Leu 50 55 60 Gln Arg Ser His Ile Val Thr Gly Asp Asp Gly His Tyr Val Ala Val 65 70 75 80 Ile Met Glu Leu Glu Ala Lys Gly Ala Lys Val Ile Pro Ile Phe Ala 85 90 95 Gly Gly Leu Asp Phe Ser Gly Pro Thr Gln Arg Tyr Leu Val Asp Pro 100 105 110 Ile Thr Gly Lys Pro Phe Val Asn Ala Val Val Ser Leu Thr Gly Phe 115 120 125 Ala Leu Val Gly Gly Pro Ala Arg Gln Asp His Pro Lys Ala Ile Ala 130 135 140 Ala Leu Gln Lys Leu Asp Val Pro Tyr Ile Val Ala Leu Pro Leu Val 145 150 155 160 Phe Gln Thr Thr Glu Glu Trp Leu Asn Ser Thr Leu Gly Leu His Pro 165 170 175 Ile Gln Val Ala Leu Gln Val Ala Leu Pro Glu Leu Asp Gly Gly Met 180 185 190 Glu Pro Ile Val Phe Ala Gly Arg Asp Pro Arg Thr Gly Lys Ser His 195 200 205 Ala Leu His Lys Arg Val Glu Gln Leu Cys Thr Arg Ala Ile Arg Trp 210 215 220 Ala Glu Leu Lys Arg Lys Thr Lys Glu Glu Lys Lys Leu Ala Ile Thr 225 230 235 240 Val Phe Ser Phe Pro Pro Asp Lys Gly Asn Val Gly Thr Ala Ala Tyr 245 250 255 Leu Asn Val Phe Asn Ser Ile Tyr Ser Val Leu Gln Asp Leu Lys Lys 260 265 270 Asp Gly Tyr Asn Val Glu Gly Leu Pro Asp Thr Ala Glu Ala Leu Ile 275 280 285 Glu Glu Val Ile His Asp Lys Glu Ala Gln Phe Asn Ser Pro Asn Leu 290 295 300 Asn Val Ala Tyr Arg Met Asn Val Arg Glu Tyr Gln Ser Leu Thr Ser 305 310 315 320 Tyr Ala Ser Leu Leu Glu Glu Asn Trp Gly Lys Pro Pro Gly Asn Leu 325 330 335 Asn Ser Asp Gly Glu Asn Leu Leu Val Tyr Gly Lys Gln Tyr Gly Asn 340 345 350 Val Phe Ile Gly Val Gln Pro Thr Phe Gly Tyr Glu Gly Asp Pro Met 355 360 365 Arg Leu Leu Phe Ser Lys Ser Ala Ser Pro His His Gly Phe Ala Ala 370 375 380 Tyr Tyr Thr Phe Val Glu Lys Ile Phe Gln Ala Asp Ala Val Leu His 385 390 395 400 Phe Gly Thr His Gly Ser Leu Glu Phe Met Pro Gly Lys Gln Val Gly 405 410 415 Met Ser Asp Ala Cys Tyr Pro Asp Ser Leu Ile Gly Asn Ile Pro Asn 420 425 430 Ile Tyr Tyr Tyr Ala Ala Asn Asn Pro Ser Glu Ala Thr Val Ala Lys 435 440 445 Arg Arg Ser Tyr Ala Asn Thr Ile Ser Tyr Leu Thr Pro Pro Ala Glu 450 455 460 Asn Ala Gly Leu Tyr Lys Gly Leu Lys Gln Leu Ser Arg Ser Ser Ser 465 470 475 480 Leu Leu Asn Gln Ser Leu Lys Asp Thr Gly Arg Gly Pro Gln Ile Val 485 490 495 Ser Ser Ile Ile Ser Thr Ala Lys Gln Cys Asn Leu Asp Lys Asp Val 500 505 510 Pro Leu Pro Glu Glu Gly Val Glu Leu Pro Pro Asn Glu Arg Asp Leu 515 520 525 Ile Val Gly Lys Val Tyr Ala Lys Ile Met Glu Ile Glu Ser Arg Leu 530 535 540 Leu Pro Cys Gly Leu His Val Ile Gly Glu Pro Pro Ser Ala Ile Glu 545 550 555 560 Ala Val Ala Thr Leu Val Asn Ile Ala Ser Leu Asp Arg Pro Glu Asp 565 570 575 Glu Ile Tyr Ser Leu Pro Asn Ile Leu Ala Gln Thr Val Gly Arg Asn 580 585 590 Ile Glu Asp Val Tyr Arg Gly Ser Asp Lys Gly Ile Leu Ala Asp Val 595 600 605 Glu Leu Leu Arg Gln Ile Thr Glu Ala Ser Arg Gly Ala Ile Thr Thr 610 615 620 Phe Val Glu Arg Thr Thr Asn Asn Lys Gly Gln Val Val Asp Val Thr 625 630 635 640 Asn Lys Leu Ser Thr Met Leu Gly Phe Gly Leu Ser Glu Pro Trp Val 645 650 655 Gln His Leu Ser Lys Thr Lys Phe Ile Arg Ala Asp Arg Glu Lys Leu 660 665 670 Arg Thr Leu Phe Thr Phe Leu Gly Glu Cys Leu Lys Leu Ile Val Ala 675 680 685 Asp Asn Glu Leu Gly Ser Leu Lys Leu Ala Leu Glu Gly Ser Tyr Val 690 695 700 Glu Pro Gly Pro Gly Gly Asp Pro Ile Arg Asn Pro Lys Val Leu Pro 705 710 715 720 Thr Gly Lys Asn Ile His Ala Leu Asp Pro Gln Ala Ile Pro Thr Thr 725 730 735 Ala Ala Leu Lys Ser Ala Lys Ile Ile Val Asp Arg Leu Leu Glu Arg 740 745 750 Gln Lys Val Asp Asn Gly Gly Lys Tyr Pro Glu Thr Ile Ala Leu Val 755 760 765 Leu Trp Gly Thr Asp Asn Ile Lys Thr Tyr Gly Glu Ser Leu Ala Gln 770 775 780 Val Leu Trp Met Ile Gly Val Arg Pro Val Ala Asp Thr Phe Gly Arg 785 790 795 800 Val Asn Arg Val Glu Pro Val Ser Leu Glu Glu Leu Gly Arg Pro Arg 805 810 815 Ile Asp Val Val Ile Asn Cys Ser Gly Val Phe Arg Asp Leu Phe Ile 820 825 830 Asn Gln Met Asn Leu Leu Asp Arg Ala Val Lys Met Val Ala Glu Leu 835 840 845 Asp Glu Pro Glu Glu Met Asn Tyr Val Arg Lys His Ala Gln Glu Gln 850 855 860 Ala Arg Glu Leu Gly Val Ser Leu Arg Glu Ala Ala Thr Arg Val Phe 865 870 875 880 Ser Asn Ala Ser Gly Ser Tyr Ser Ser Asn Val Asn Leu Ala Val Glu 885 890 895 Asn Ala Ser Trp Thr Asp Glu Lys Gln Leu Gln Asp Met Tyr Leu Ser 900 905 910 Arg Lys Ser Phe Ala Phe Asp Cys Asp Ala Pro Gly Ala Gly Met Arg 915 920 925 Glu Gln Arg Lys Thr Phe Glu Leu Ala Leu Ala Thr Ala Asp Ala Thr 930 935 940 Phe Gln Asn Leu Asp Ser Ser Glu Ile Ser Leu Thr Asp Val Ser His 945 950 955 960 Tyr Phe Asp Ser Asp Pro Thr Lys Leu Val Gln Gly Leu Arg Lys Asp 965 970 975 Gly Arg Ala Pro Ser Ser Tyr Ile Ala Asp Thr Thr Thr Ala Asn Ala 980 985 990 Gln Val Arg Thr Leu Ser Glu Thr Val Arg Leu Asp Ala Arg Thr Lys 995 1000 1005 Leu Leu Asn Pro Lys Trp Tyr Glu Gly Met Met Lys Ser Gly Tyr Glu 1010 1015 1020 Gly Val Arg Glu Ile Glu Lys Arg Leu Thr Asn Thr Val Gly Trp Ser 1025 1030 1035 1040 Ala Thr Ser Gly Gln Val Asp Asn Trp Val Tyr Glu Glu Ala Asn Ala 1045 1050 1055 Thr Phe Ile Glu Asp Glu Ala Met Arg Lys Arg Leu Met Asp Thr Asn 1060 1065 1070 Pro Asn Ser Phe Arg Lys Leu Val Gln Thr Phe Leu Glu Ala Ser Gly 1075 1080 1085 Arg Gly Tyr Trp Glu Thr Ser Glu Glu Asn Leu Glu Lys Leu Arg Glu 1090 1095 1100 Leu Tyr Ser Glu Val Glu Asp Lys Ile Glu Gly Ile Asp Arg 1105 1110 1115 37 511 DNA Triticum aestivum unsure (317) n is a, c, g or t 37 ttttttttta cattcacaac gaaccaccct ctttgttaaa ggtctgtatg gcatgttaca 60 acttttataa atcacaaatt atacaagttt cgactcagca aggcagaaga atatttcttc 120 ttggtcaaat gctggaactg gttgatcagt gaaatgagtt caccggtcaa ttccttcgat 180 cttgtcttca acctccgagt agagctccct gagcctttcc aagttatcct ctgatgtctc 240 ccaagtagcc cctgccattt gcttctagga aggttgaagc atttcctgaa cgaatggggg 300 tggtgtccat cagctcntcc tcatctcctc ancctcaatg aatgtggtat tgctcctcgt 360 aaaccagttg tccaactgcc cngatgttgc acccaaccaa cantattggc aancnttcnc 420 attcccctac tccctcaaag catcctcaac atccctcgta caactaggtc natactttgg 480 cccttgcacn aagagacgtc tccgaaagtc g 511 38 76 PRT Triticum aestivum UNSURE (20) Xaa can be any naturally occurring amino acid 38 Ala Thr Ser Gly Gln Leu Asp Asn Trp Phe Thr Arg Ser Asn Thr Thr 1 5 10 15 Phe Ile Glu Xaa Glu Glu Met Arg Xaa Ser Xaa Trp Thr Pro Pro Pro 20 25 30 Phe Val Gln Glu Met Leu Gln Pro Ser Xaa Lys Gln Met Ala Gly Ala 35 40 45 Thr Trp Glu Thr Ser Glu Asp Asn Leu Glu Arg Leu Arg Glu Leu Tyr 50 55 60 Ser Glu Val Glu Asp Lys Ile Glu Gly Ile Asp Arg 65 70 75 39 3257 DNA Triticum aestivum 39 ccaggggcgc caaggtcatc cccatcttcg ccggcgggct cgacttctcc ggccccatcg 60 agcgctacct cgtcgacccc atcaccaaga agccgttcgt gaacgccgtg gtgtcgctca 120 ccgggttcgc gctcgtcggc gggccggcca ggcaggacca ccccaaggcc atcgcctcgc 180 tgatgaagct agacgtgccg tacatcgtcg cgctgccgct cgtgttccag accacggagg 240 agtggctcaa cagcaccttg ggccttcacc ccatccaggt ggcgctgcag gttgcgctcc 300 cggagctcga cggcggcatg gagcccatcg tgttcgccgg ccgggacccg agatcaggga 360 agtcgcatgc attgcacaag agggtggagc agctctgcac tagagcgatc agatgggcag 420 aactcaagag gaaaactaag atggacaaga aactagccat caccgttttc agcttcccac 480 cagacaaggg caatgtcggc actgcagcat acctgaatgt cttcagttcc atctattctg 540 tcctcaagga tctcaagaag gatggctaca atgtcgaggg tcttccagag acacctgaag 600 aactcattga ggaggttatt catgataagg aggcccagtt caacagcccc aacctcaatg 660 ttgtttaccg catgaatgtg cgggagtacc aagcactcac cccctacgcc aacatgctgg 720 aggagaactg gggcaagcca cctgggcatc tcaactctga tggtgagaac ctccttgtct 780 atgggaagca gtatggcaac atcttcatcg gagtgcagcc cacttttggc tatgaaggtg 840 atccaatgcg gcttctgttc tcaaagtctg ccagccctca ccatgggttt gcggcatact 900 acacttttgt tgagaagatc ttcaaggcag atgctgttct gcacttcggc acacacgggt 960 cccttgagtt catgcccggg aaacaagttg gaatgagtga tgcatgcttc cctgatagtc 1020 tgattggtaa catccccaac atctactact atgcagcaaa caacccttca gaggcaacag 1080 tggccaagcg tcgaagctat gcaaacacca taagctacct gacaccacca gctgagaatg 1140 ccggtctgta caaggggctg aagcagctgt cagagctcat cgcctcttac cagtcactca 1200 aggacacggg ccgtggcaac cagattgtga gctcaatcat cagcactgca aaacagtgta 1260 acctggacaa ggatgttgac ttgcccgatg aaggcgagga gctcccagcc aatgagcgtg 1320 acctcgtcgt cgggaaggtg tatggaaagc tcatggagat agagtcacgg ctactgccat 1380 gtggtctgca tgtgataggt gagccaccaa ctgctgtcga agctgtggcc acattggtga 1440 acatagctgc ccttgaccgc ccagaggaga acatattctc gctgcccggc attcttgctg 1500 cgacggtggg caggaccatt gaagatgtgt acaggggtag cgacaagggc atactggctg 1560 atgttgaact cctgaagcag atcactgaag cttcacgagg tgccgtaggt gcctttgttg 1620 agaagactac aaacagcaaa gggcaagttg ttgatgttaa aagcaaactc agttccatcc 1680 ttggctttgg tctctcagag ccatgggtgg agtacctgtc ccagaccaag ttcatcaggg 1740 cggacagaga taagctgagg accttgtttg gattcttggg agagtgcctg aagctgattg 1800 tggcagacaa tgagctggga gccttgaaga ctgcccttga gggaagctat gttgagcctg 1860 gccctggtgg tgatcccatc cgtaacccaa aggttctccc aacagggaag aacatccatg 1920 ctctcgaccc gcagtctatc ccgactgcag ctgccatgaa gagtgccaag attgttgtgg 1980 aacgtctgct ggagcggcaa aaggctgaca atggtggcaa gtatcctgag acaattgcac 2040 ttgtcttgtg gggcaccgac aacatcaaga cctacggcga gtcactggcc caggtgatgt 2100 ggatgcttgg tgtggagccg gttactgatg ggcttggccg tgtcaaccgt gtggagcccg 2160 tcagcattga ggagcttgga cgccctagga ttgatgtcgt cgtcaactgc tcgggtgtgt 2220 tcagagatct tttcatcaac cagatgaatc tgctggaccg ggcagtaaag atggttgctg 2280 aactggatga gccaattgag atgaactatg tgcgcaagca tgcccaggag caggcagagg 2340 agctcggtgt ctcggtaaga gaggcggcaa caaggatctt ctcaaatgca tcaggctctt 2400 actcgtcgaa tgtgaacttg gcagtggaga atgcatcatg gacagatgag aagcagctcc 2460 aggacatgta cctgagccgc aagtcttttg cgttcgacag tgatgctcca ggggtaggca 2520 tgctagagaa acgcaagacg tttgagcttg ctctagcaac agcagatgcc acattccaaa 2580 acctggactc gtcggagatc tcactgacgg atgtcagcca ctacttcgac tcagacccga 2640 cgaagctggt gcaagggctg cggaaggatg ggcgggcacc ttcatcgtac atagcagaca 2700 caaccacggc aaatgcacag gtgcggacat tgtcggagac ggtgcgtctt gatgcaagga 2760 caaagctact gaaccctagg tggtacgagg ggatgatgaa gagtggctat gagggagtta 2820 gagagatcga gaagcggctg acaaatactg ttggatggag tgcaacatcc gggcaggtgg 2880 acaactgggt ttacgaggaa gcaaatacca cattcattga agatgaagag atgaggaaga 2940 ggctgatgga caccaacccc aattcgttca ggaaactgct tcaaaccttc ctagaagcaa 3000 atggcagggg ctactgggag acatcagagg ataacttgga aaggctcagg gagctctact 3060 cggaggttga agacaagatc gaaggaattg accggtgaac tcatttcact gatcaaccag 3120 ttccagcatt tgaccaagaa gaaatattct tctgccttgc tgagtcgaaa cttgtataat 3180 ttgtgattta taaaagttgt aacatgccat acagaccttt aacaaagagg gtggttcgtt 3240 gtgaatgtaa aaaaaaa 3257 40 1031 PRT Triticum aestivum 40 Arg Gly Ala Lys Val Ile Pro Ile Phe Ala Gly Gly Leu Asp Phe Ser 1 5 10 15 Gly Pro Ile Glu Arg Tyr Leu Val Asp Pro Ile Thr Lys Lys Pro Phe 20 25 30 Val Asn Ala Val Val Ser Leu Thr Gly Phe Ala Leu Val Gly Gly Pro 35 40 45 Ala Arg Gln Asp His Pro Lys Ala Ile Ala Ser Leu Met Lys Leu Asp 50 55 60 Val Pro Tyr Ile Val Ala Leu Pro Leu Val Phe Gln Thr Thr Glu Glu 65 70 75 80 Trp Leu Asn Ser Thr Leu Gly Leu His Pro Ile Gln Val Ala Leu Gln 85 90 95 Val Ala Leu Pro Glu Leu Asp Gly Gly Met Glu Pro Ile Val Phe Ala 100 105 110 Gly Arg Asp Pro Arg Ser Gly Lys Ser His Ala Leu His Lys Arg Val 115 120 125 Glu Gln Leu Cys Thr Arg Ala Ile Arg Trp Ala Glu Leu Lys Arg Lys 130 135 140 Thr Lys Met Asp Lys Lys Leu Ala Ile Thr Val Phe Ser Phe Pro Pro 145 150 155 160 Asp Lys Gly Asn Val Gly Thr Ala Ala Tyr Leu Asn Val Phe Ser Ser 165 170 175 Ile Tyr Ser Val Leu Lys Asp Leu Lys Lys Asp Gly Tyr Asn Val Glu 180 185 190 Gly Leu Pro Glu Thr Pro Glu Glu Leu Ile Glu Glu Val Ile His Asp 195 200 205 Lys Glu Ala Gln Phe Asn Ser Pro Asn Leu Asn Val Val Tyr Arg Met 210 215 220 Asn Val Arg Glu Tyr Gln Ala Leu Thr Pro Tyr Ala Asn Met Leu Glu 225 230 235 240 Glu Asn Trp Gly Lys Pro Pro Gly His Leu Asn Ser Asp Gly Glu Asn 245 250 255 Leu Leu Val Tyr Gly Lys Gln Tyr Gly Asn Ile Phe Ile Gly Val Gln 260 265 270 Pro Thr Phe Gly Tyr Glu Gly Asp Pro Met Arg Leu Leu Phe Ser Lys 275 280 285 Ser Ala Ser Pro His His Gly Phe Ala Ala Tyr Tyr Thr Phe Val Glu 290 295 300 Lys Ile Phe Lys Ala Asp Ala Val Leu His Phe Gly Thr His Gly Ser 305 310 315 320 Leu Glu Phe Met Pro Gly Lys Gln Val Gly Met Ser Asp Ala Cys Phe 325 330 335 Pro Asp Ser Leu Ile Gly Asn Ile Pro Asn Ile Tyr Tyr Tyr Ala Ala 340 345 350 Asn Asn Pro Ser Glu Ala Thr Val Ala Lys Arg Arg Ser Tyr Ala Asn 355 360 365 Thr Ile Ser Tyr Leu Thr Pro Pro Ala Glu Asn Ala Gly Leu Tyr Lys 370 375 380 Gly Leu Lys Gln Leu Ser Glu Leu Ile Ala Ser Tyr Gln Ser Leu Lys 385 390 395 400 Asp Thr Gly Arg Gly Asn Gln Ile Val Ser Ser Ile Ile Ser Thr Ala 405 410 415 Lys Gln Cys Asn Leu Asp Lys Asp Val Asp Leu Pro Asp Glu Gly Glu 420 425 430 Glu Leu Pro Ala Asn Glu Arg Asp Leu Val Val Gly Lys Val Tyr Gly 435 440 445 Lys Leu Met Glu Ile Glu Ser Arg Leu Leu Pro Cys Gly Leu His Val 450 455 460 Ile Gly Glu Pro Pro Thr Ala Val Glu Ala Val Ala Thr Leu Val Asn 465 470 475 480 Ile Ala Ala Leu Asp Arg Pro Glu Glu Asn Ile Phe Ser Leu Pro Gly 485 490 495 Ile Leu Ala Ala Thr Val Gly Arg Thr Ile Glu Asp Val Tyr Arg Gly 500 505 510 Ser Asp Lys Gly Ile Leu Ala Asp Val Glu Leu Leu Lys Gln Ile Thr 515 520 525 Glu Ala Ser Arg Gly Ala Val Gly Ala Phe Val Glu Lys Thr Thr Asn 530 535 540 Ser Lys Gly Gln Val Val Asp Val Lys Ser Lys Leu Ser Ser Ile Leu 545 550 555 560 Gly Phe Gly Leu Ser Glu Pro Trp Val Glu Tyr Leu Ser Gln Thr Lys 565 570 575 Phe Ile Arg Ala Asp Arg Asp Lys Leu Arg Thr Leu Phe Gly Phe Leu 580 585 590 Gly Glu Cys Leu Lys Leu Ile Val Ala Asp Asn Glu Leu Gly Ala Leu 595 600 605 Lys Thr Ala Leu Glu Gly Ser Tyr Val Glu Pro Gly Pro Gly Gly Asp 610 615 620 Pro Ile Arg Asn Pro Lys Val Leu Pro Thr Gly Lys Asn Ile His Ala 625 630 635 640 Leu Asp Pro Gln Ser Ile Pro Thr Ala Ala Ala Met Lys Ser Ala Lys 645 650 655 Ile Val Val Glu Arg Leu Leu Glu Arg Gln Lys Ala Asp Asn Gly Gly 660 665 670 Lys Tyr Pro Glu Thr Ile Ala Leu Val Leu Trp Gly Thr Asp Asn Ile 675 680 685 Lys Thr Tyr Gly Glu Ser Leu Ala Gln Val Met Trp Met Leu Gly Val 690 695 700 Glu Pro Val Thr Asp Gly Leu Gly Arg Val Asn Arg Val Glu Pro Val 705 710 715 720 Ser Ile Glu Glu Leu Gly Arg Pro Arg Ile Asp Val Val Val Asn Cys 725 730 735 Ser Gly Val Phe Arg Asp Leu Phe Ile Asn Gln Met Asn Leu Leu Asp 740 745 750 Arg Ala Val Lys Met Val Ala Glu Leu Asp Glu Pro Ile Glu Met Asn 755 760 765 Tyr Val Arg Lys His Ala Gln Glu Gln Ala Glu Glu Leu Gly Val Ser 770 775 780 Val Arg Glu Ala Ala Thr Arg Ile Phe Ser Asn Ala Ser Gly Ser Tyr 785 790 795 800 Ser Ser Asn Val Asn Leu Ala Val Glu Asn Ala Ser Trp Thr Asp Glu 805 810 815 Lys Gln Leu Gln Asp Met Tyr Leu Ser Arg Lys Ser Phe Ala Phe Asp 820 825 830 Ser Asp Ala Pro Gly Val Gly Met Leu Glu Lys Arg Lys Thr Phe Glu 835 840 845 Leu Ala Leu Ala Thr Ala Asp Ala Thr Phe Gln Asn Leu Asp Ser Ser 850 855 860 Glu Ile Ser Leu Thr Asp Val Ser His Tyr Phe Asp Ser Asp Pro Thr 865 870 875 880 Lys Leu Val Gln Gly Leu Arg Lys Asp Gly Arg Ala Pro Ser Ser Tyr 885 890 895 Ile Ala Asp Thr Thr Thr Ala Asn Ala Gln Val Arg Thr Leu Ser Glu 900 905 910 Thr Val Arg Leu Asp Ala Arg Thr Lys Leu Leu Asn Pro Arg Trp Tyr 915 920 925 Glu Gly Met Met Lys Ser Gly Tyr Glu Gly Val Arg Glu Ile Glu Lys 930 935 940 Arg Leu Thr Asn Thr Val Gly Trp Ser Ala Thr Ser Gly Gln Val Asp 945 950 955 960 Asn Trp Val Tyr Glu Glu Ala Asn Thr Thr Phe Ile Glu Asp Glu Glu 965 970 975 Met Arg Lys Arg Leu Met Asp Thr Asn Pro Asn Ser Phe Arg Lys Leu 980 985 990 Leu Gln Thr Phe Leu Glu Ala Asn Gly Arg Gly Tyr Trp Glu Thr Ser 995 1000 1005 Glu Asp Asn Leu Glu Arg Leu Arg Glu Leu Tyr Ser Glu Val Glu Asp 1010 1015 1020 Lys Ile Glu Gly Ile Asp Arg 1025 1030 41 466 DNA Zea mays unsure (14) n is a, c, g or t 41 ctcctccccg tcanggcttc naccttctcc cccacttccg ccgcgagggc cctcctcccg 60 ggctccacct cccgcccact cttcctcgcc gcttcagctt cctcagggcg cattcaacca 120 tccaggaagg gactggactt ccgccgcggc cgattcaccg ttctgaaatt cgccgctccc 180 accgccgccg aacaggaggc gacggcgtcg gncgcgaagg agacccagcg ccccgtgtac 240 ccgttcgcgg ccatcgtggg gcaggacgag atgaagctct gcctgctgct caacgtcatc 300 gaccccaaga tcggcggcgt catgatcatg ggcgaacagg ggggaacggg gaaatccaac 360 aacggttcgg ctnccntccg tcgaacctgg ctncccgggt aatccgcctt cggncngtcg 420 ggggaacccc tttaaactcc ngaacccggg tngaancccg gangtg 466 42 66 PRT Zea mays UNSURE (23) Xaa can be any naturally occurring amino acid 42 Arg Gly Arg Phe Thr Val Leu Lys Phe Ala Ala Pro Thr Ala Ala Glu 1 5 10 15 Gln Glu Ala Thr Ala Ser Xaa Ala Lys Glu Thr Gln Arg Pro Val Tyr 20 25 30 Pro Phe Ala Ala Ile Val Gly Gln Asp Glu Met Lys Leu Cys Leu Leu 35 40 45 Leu Asn Val Ile Asp Pro Lys Ile Gly Gly Val Met Ile Met Gly Glu 50 55 60 Gln Gly 65 43 1377 DNA Zea mays 43 ccacgcgtcc gctcctcccc gtcatggctt ccaccttctc ccccacttcc gccgcgaggg 60 ccctcctccc gggctccacc tcccgcccac tcttcctcgc cgcttcagct tcctcagggc 120 gcattcaacc atccaggaag ggactggact tccgccgcgg ccgattcacc gtctgcaatg 180 tcgccgctcc caccgccgcc gaacaggagg cgacggcgtc ggccgcgaag gagacccagc 240 gccccgtgta cccgttcgcg gccatcgtgg ggcaggacga gatgaagctc tgcctgctgc 300 tcaacgtcat cgaccccaag atcggcggcg tcatgatcat gggcgacagg ggcacgggga 360 agtccaccac cgtccgctcc ctcgtcgacc tgctcccgga catccgcgtc gtcgtcggcg 420 accccttcaa ctccgacccg gacgaccccg aggtcatggg ccccgaggtc cgccaacggg 480 tcctgcaggg ggacaccggc ctccccgtca ccaccgccat agtcaccatg gtcgacctgc 540 ccctgggcgc caccgaggac cgcgtctgcg gcaccattga catcgagaag gcgctcaccg 600 agggcgtcaa ggcgttcgag cccggcctgc tcgccaaggc caacaggggc atactgtacg 660 tcgacgaggt caacctgctg gacgaccacc tcgtcgacgt gctgctggat tccgctgcgt 720 cggggtggaa cacggtggag agggagggta tctccatatc ccaccttgtt ggctttatct 780 taatgggttt tgttaacccg gaggaggggg agttcagccc ccagttgttg gaccggttcg 840 ggttgcaggc ccaggttgtt ccgttcaggg acccggagtt caggttgaaa atcttggagg 900 ggaggcttgt tttcgacagg aatccgaaga cgttccgtga gtcgtatcat gacgagcagg 960 agaagctcca gcagcagata tcatctgcac ggagtaacct tggcgctgtg cagattgacc 1020 atgacctccg tgtcaagata tccaaggtgt gctctgagtt gaacgttgat ggactcagag 1080 gtgacattgt gactaacagg gctgccaagg cgctggctgc gttgaaagga agggacagcg 1140 tcaccgtgga ggacattgct actgtcattc caaactgctt gaggcatcgg ctccgcaagg 1200 atccgcttga atccattgac tcgggtttac ttgtcattga gaagttttat gaagtcttta 1260 gctagattgt tcttgaggta aatgttcctt tgtcacaatt tttggcggga accctcttgt 1320 tctgttactt tcataatgtt ctgctgttta ataatatctg gagcttgaat tggtatc 1377 44 413 PRT Zea mays 44 Met Ala Ser Thr Phe Ser Pro Thr Ser Ala Ala Arg Ala Leu Leu Pro 1 5 10 15 Gly Ser Thr Ser Arg Pro Leu Phe Leu Ala Ala Ser Ala Ser Ser Gly 20 25 30 Arg Ile Gln Pro Ser Arg Lys Gly Leu Asp Phe Arg Arg Gly Arg Phe 35 40 45 Thr Val Cys Asn Val Ala Ala Pro Thr Ala Ala Glu Gln Glu Ala Thr 50 55 60 Ala Ser Ala Ala Lys Glu Thr Gln Arg Pro Val Tyr Pro Phe Ala Ala 65 70 75 80 Ile Val Gly Gln Asp Glu Met Lys Leu Cys Leu Leu Leu Asn Val Ile 85 90 95 Asp Pro Lys Ile Gly Gly Val Met Ile Met Gly Asp Arg Gly Thr Gly 100 105 110 Lys Ser Thr Thr Val Arg Ser Leu Val Asp Leu Leu Pro Asp Ile Arg 115 120 125 Val Val Val Gly Asp Pro Phe Asn Ser Asp Pro Asp Asp Pro Glu Val 130 135 140 Met Gly Pro Glu Val Arg Gln Arg Val Leu Gln Gly Asp Thr Gly Leu 145 150 155 160 Pro Val Thr Thr Ala Ile Val Thr Met Val Asp Leu Pro Leu Gly Ala 165 170 175 Thr Glu Asp Arg Val Cys Gly Thr Ile Asp Ile Glu Lys Ala Leu Thr 180 185 190 Glu Gly Val Lys Ala Phe Glu Pro Gly Leu Leu Ala Lys Ala Asn Arg 195 200 205 Gly Ile Leu Tyr Val Asp Glu Val Asn Leu Leu Asp Asp His Leu Val 210 215 220 Asp Val Leu Leu Asp Ser Ala Ala Ser Gly Trp Asn Thr Val Glu Arg 225 230 235 240 Glu Gly Ile Ser Ile Ser His Leu Val Gly Phe Ile Leu Met Gly Phe 245 250 255 Val Asn Pro Glu Glu Gly Glu Phe Ser Pro Gln Leu Leu Asp Arg Phe 260 265 270 Gly Leu Gln Ala Gln Val Val Pro Phe Arg Asp Pro Glu Phe Arg Leu 275 280 285 Lys Ile Leu Glu Gly Arg Leu Val Phe Asp Arg Asn Pro Lys Thr Phe 290 295 300 Arg Glu Ser Tyr His Asp Glu Gln Glu Lys Leu Gln Gln Gln Ile Ser 305 310 315 320 Ser Ala Arg Ser Asn Leu Gly Ala Val Gln Ile Asp His Asp Leu Arg 325 330 335 Val Lys Ile Ser Lys Val Cys Ser Glu Leu Asn Val Asp Gly Leu Arg 340 345 350 Gly Asp Ile Val Thr Asn Arg Ala Ala Lys Ala Leu Ala Ala Leu Lys 355 360 365 Gly Arg Asp Ser Val Thr Val Glu Asp Ile Ala Thr Val Ile Pro Asn 370 375 380 Cys Leu Arg His Arg Leu Arg Lys Asp Pro Leu Glu Ser Ile Asp Ser 385 390 395 400 Gly Leu Leu Val Ile Glu Lys Phe Tyr Glu Val Phe Ser 405 410 45 602 DNA Oryza sativa unsure (345) n is a, c, g or t 45 tacacacacc attgatattg agaaggcgct caccgatggt gtcaaggcgt tcgagcctgg 60 tttgcttgcc aaggccaaca gggggattct ttatgtggat gaggtcaatt tgttggatga 120 ccatctagta gatgtgcttc tggattctgc tgcgtcagga tggaacaccg tggagagaga 180 gggtatctcc atctcccacc ctgctcggtt catcctcatt gggtctgggt aaccccgagg 240 aaggggagct ccggccacag ctgcttgacc ggtttggcat gcacgcgcag ttggtactgt 300 cagggatgct gaactcaagg gtgaaaatgt tgaagagaga ctccngtcga cagggattcc 360 aaagcttccg ttgatcctac tttggaggaa caagacaact ccaacagcaa attcaaccgc 420 tccgataacc ttggtgctgt gcaaattgac caatgatctt cntgttaaga ttccaaatgt 480 gtgcaaattn aatgttnatg gattaanang ggacatnttg actaacaagg ctgcaaagng 540 ttngnaacac caaangnang gacacttcac tgtnaaggac attgcactgt tttcccaact 600 gc 602 46 74 PRT Oryza sativa 46 Thr Ile Asp Ile Glu Lys Ala Leu Thr Asp Gly Val Lys Ala Phe Glu 1 5 10 15 Pro Gly Leu Leu Ala Lys Ala Asn Arg Gly Ile Leu Tyr Val Asp Glu 20 25 30 Val Asn Leu Leu Asp Asp His Leu Val Asp Val Leu Leu Asp Ser Ala 35 40 45 Ala Ser Gly Trp Asn Thr Val Glu Arg Glu Gly Ile Ser Ile Ser His 50 55 60 Pro Ala Arg Phe Ile Leu Ile Gly Ser Gly 65 70 47 463 DNA Oryza sativa unsure (200) n is a, c, g or t 47 tgtatcctcc tcaccatggc ttccgccttc tcccccgcca ccgccgcgcc cgccgcgtcg 60 ccggccctct tctccgcctc cacctcccgg cctctctccc tcaccgccgc cgccgctgcc 120 gtctcagccc gtatcccgtc acggagaggg ttccgccgcg gccgcttcac cgtctgcaat 180 gtagccgccc cctccgccan ccagcaggag gctaaggcgg cgggcgcgaa ggagagccaa 240 cggccggtgt atccgttcgc ggcgatcgtg gggcaggacg agatgaagct gtgcctgntg 300 ctcnacgtca tcgaccctaa gatcggcggt gtcatgatca tgggagancg tgcaccggca 360 aatccaacaa cgtccgtcgn tcgtcgaant gntcccggat atcgcgtcgt tgttgggaac 420 ctttaaatcc gancctanga ttccgaggta tnggncntga ggc 463 48 72 PRT Oryza sativa UNSURE (15) Xaa can be any naturally occurring amino acid 48 Arg Gly Arg Phe Thr Val Cys Asn Val Ala Ala Pro Ser Ala Xaa Gln 1 5 10 15 Gln Glu Ala Lys Ala Ala Gly Ala Lys Glu Ser Gln Arg Pro Val Tyr 20 25 30 Pro Phe Ala Ala Ile Val Gly Gln Asp Glu Met Lys Leu Cys Leu Xaa 35 40 45 Leu Xaa Val Ile Asp Pro Lys Ile Gly Gly Val Met Ile Met Gly Xaa 50 55 60 Arg Ala Pro Ala Asn Pro Thr Thr 65 70 49 1408 DNA Oryza sativa 49 gcacgagtgt atcctcctca ccatggcttc cgccttctcc cccgccaccg ccgcgcccgc 60 cgcgtcgccg gccctcttct ccgcctccac ctcccggcct ctctccctca ccgccgccgc 120 cgctgccgtc tcagcccgta tcccgtcacg gagagggttc cgccgcggcc gcttcaccgt 180 ctgcaatgta gccgccccct ccgccaccca gcaggaggct aaggcggcgg gcgcgaagga 240 gagccaacgg ccggtgtatc cgttcgcggc gatcgtgggg caggacgaga tgaagctgtg 300 cctgctgctc aacgtcatcg accctaagat cggcggtgtc atgatcatgg gagaccgtgg 360 caccggcaaa tccaccaccg tccgctcgct cgtcgacctg ctcccggata tccgcgtcgt 420 tgttggcgac cctttcaatt ccgaccctga cgatcccgag gtcatgggcc ctgaggtccg 480 ggaacgcgtg ctggagggtg agaagcttcc tgttgtcacg gccaagatca ccatggtaga 540 tcttcccctt ggtgccactg aggatagagt ctgtggcacc attgatattg agaaggcgct 600 caccgatggt gtcaaggcgt tcgagcctgg tttgcttgcc aaggccaaca gggggattct 660 ttatgtggat gaggtcaatt tgttggatga ccatctagta gatgtgcttc tggattctgc 720 tgcgtcagga tggaacaccg tggagagaga gggtatctcc atctcccacc ctgctcggtt 780 catcctcatt gggtctggta accccgagga aggggagctc cggccacagc tgcttgaccg 840 gtttggcatg cacgcgcagg ttggtactgt cagggatgct gaactcaggg tgaaaattgt 900 tgaagagaga gctcggttcg acagggatcc aaaggcgttc cgtgagtcct acttggagga 960 acaagacaag ctccagcagc agatttcatc tgctcggagt aaccttggtg ctgtgcagat 1020 tgaccatgat cttcgtgtta agatttctaa agtgtgtgca gagttgaatg ttgatggatt 1080 aagaggggac attgtgacta acagggctgc caaggcgttg gcagcactca aaggcaggga 1140 cactgtcact gtagaggaca ttgccactgt tatccccaac tgcttgaggc atcggcttcg 1200 gaaggaccca cttgaatcaa ttgactcagg attgctcgtg gttgagaagt tttatgaagt 1260 cttcacctaa attattctgg aggtaaatgg ttttctatca gaaagttcgg caggagggct 1320 tttgtttgag tttaatgaca ttgtttcaga ggcttgaact tgatgtctat ttgtacatct 1380 atcattagta tagattttat tccccttc 1408 50 415 PRT Oryza sativa 50 Met Ala Ser Ala Phe Ser Pro Ala Thr Ala Ala Pro Ala Ala Ser Pro 1 5 10 15 Ala Leu Phe Ser Ala Ser Thr Ser Arg Pro Leu Ser Leu Thr Ala Ala 20 25 30 Ala Ala Ala Val Ser Ala Arg Ile Pro Ser Arg Arg Gly Phe Arg Arg 35 40 45 Gly Arg Phe Thr Val Cys Asn Val Ala Ala Pro Ser Ala Thr Gln Gln 50 55 60 Glu Ala Lys Ala Ala Gly Ala Lys Glu Ser Gln Arg Pro Val Tyr Pro 65 70 75 80 Phe Ala Ala Ile Val Gly Gln Asp Glu Met Lys Leu Cys Leu Leu Leu 85 90 95 Asn Val Ile Asp Pro Lys Ile Gly Gly Val Met Ile Met Gly Asp Arg 100 105 110 Gly Thr Gly Lys Ser Thr Thr Val Arg Ser Leu Val Asp Leu Leu Pro 115 120 125 Asp Ile Arg Val Val Val Gly Asp Pro Phe Asn Ser Asp Pro Asp Asp 130 135 140 Pro Glu Val Met Gly Pro Glu Val Arg Glu Arg Val Leu Glu Gly Glu 145 150 155 160 Lys Leu Pro Val Val Thr Ala Lys Ile Thr Met Val Asp Leu Pro Leu 165 170 175 Gly Ala Thr Glu Asp Arg Val Cys Gly Thr Ile Asp Ile Glu Lys Ala 180 185 190 Leu Thr Asp Gly Val Lys Ala Phe Glu Pro Gly Leu Leu Ala Lys Ala 195 200 205 Asn Arg Gly Ile Leu Tyr Val Asp Glu Val Asn Leu Leu Asp Asp His 210 215 220 Leu Val Asp Val Leu Leu Asp Ser Ala Ala Ser Gly Trp Asn Thr Val 225 230 235 240 Glu Arg Glu Gly Ile Ser Ile Ser His Pro Ala Arg Phe Ile Leu Ile 245 250 255 Gly Ser Gly Asn Pro Glu Glu Gly Glu Leu Arg Pro Gln Leu Leu Asp 260 265 270 Arg Phe Gly Met His Ala Gln Val Gly Thr Val Arg Asp Ala Glu Leu 275 280 285 Arg Val Lys Ile Val Glu Glu Arg Ala Arg Phe Asp Arg Asp Pro Lys 290 295 300 Ala Phe Arg Glu Ser Tyr Leu Glu Glu Gln Asp Lys Leu Gln Gln Gln 305 310 315 320 Ile Ser Ser Ala Arg Ser Asn Leu Gly Ala Val Gln Ile Asp His Asp 325 330 335 Leu Arg Val Lys Ile Ser Lys Val Cys Ala Glu Leu Asn Val Asp Gly 340 345 350 Leu Arg Gly Asp Ile Val Thr Asn Arg Ala Ala Lys Ala Leu Ala Ala 355 360 365 Leu Lys Gly Arg Asp Thr Val Thr Val Glu Asp Ile Ala Thr Val Ile 370 375 380 Pro Asn Cys Leu Arg His Arg Leu Arg Lys Asp Pro Leu Glu Ser Ile 385 390 395 400 Asp Ser Gly Leu Leu Val Val Glu Lys Phe Tyr Glu Val Phe Thr 405 410 415 51 754 PRT Pisum sativum 51 Met Gly Phe Ser Leu Thr His Thr Pro His Thr Thr Ala Ser Pro Asn 1 5 10 15 Leu Gln Leu Arg Phe His Ser Leu Leu Pro Pro Ser Phe Thr Ser Gln 20 25 30 Pro Phe Leu Ser Leu His Ser Thr Phe Pro Pro Lys Arg Thr Val Pro 35 40 45 Lys Leu Arg Ala Gln Ser Glu Asn Gly Ala Val Leu Gln Ala Ser Glu 50 55 60 Glu Lys Leu Asp Ala Ser Asn Tyr Gly Arg Gln Tyr Phe Pro Leu Ala 65 70 75 80 Ala Val Ile Gly Gln Asp Ala Ile Lys Thr Ala Leu Leu Leu Gly Ala 85 90 95 Thr Asp Pro Arg Ile Gly Gly Ile Ala Ile Ser Gly Arg Arg Gly Thr 100 105 110 Ala Lys Thr Ile Met Ala Arg Gly Met His Ala Ile Leu Pro Pro Ile 115 120 125 Glu Val Val Gln Gly Ser Ile Ala Asn Ala Asp Pro Ser Cys Pro Glu 130 135 140 Glu Trp Glu Asp Gly Leu Tyr Lys Arg Val Glu Tyr Asp Ser Asp Gly 145 150 155 160 Asn Val Lys Thr His Ile Ile Lys Ser Pro Phe Val Gln Ile Pro Leu 165 170 175 Gly Val Thr Glu Asp Arg Leu Ile Gly Ser Val Asp Val Glu Glu Ser 180 185 190 Val Lys Thr Gly Thr Thr Val Phe Gln Pro Gly Leu Leu Ala Glu Ala 195 200 205 His Arg Gly Val Leu Tyr Val Asp Glu Ile Asn Leu Leu Asp Glu Gly 210 215 220 Ile Ser Asn Leu Leu Leu Asn Val Leu Thr Glu Gly Val Asn Ile Val 225 230 235 240 Glu Arg Glu Gly Ile Ser Phe Arg His Pro Cys Arg Pro Leu Leu Ile 245 250 255 Ala Thr Tyr Asn Pro Asp Glu Gly Ser Val Arg Glu His Leu Leu Asp 260 265 270 Arg Ile Ala Ile Asn Leu Ser Ala Asp Leu Pro Met Ser Phe Glu Asn 275 280 285 Arg Val Glu Ala Val Gly Ile Ala Thr Glu Phe Gln Asp Asn Cys Gly 290 295 300 Gln Val Phe Lys Met Val Asp Glu Asp Thr Asp Asn Ala Lys Thr Gln 305 310 315 320 Ile Ile Leu Ala Arg Glu Tyr Leu Lys Asp Val Thr Ile Ser Lys Glu 325 330 335 Gln Leu Lys Tyr Leu Val Ile Glu Ala Leu Arg Gly Gly Val Gln Gly 340 345 350 His Arg Ala Glu Leu Tyr Ala Ala Arg Val Ala Lys Cys Leu Ala Ala 355 360 365 Leu Glu Gly Arg Glu Lys Val Tyr Val Asp Asp Leu Lys Lys Ala Val 370 375 380 Glu Leu Val Ile Leu Pro Arg Ser Ile Ile Thr Asp Thr Pro Pro Glu 385 390 395 400 Gln Gln Asn Gln Pro Pro Pro Pro Pro Pro Pro Pro Gln Asn Gln Glu 405 410 415 Ser Asn Glu Glu Gln Asn Glu Glu Glu Glu Gln Glu Glu Glu Glu Glu 420 425 430 Asp Asp Asn Asp Glu Glu Asn Glu Gln Gln Gln Asp Gln Leu Pro Glu 435 440 445 Glu Phe Ile Phe Asp Ala Glu Gly Gly Leu Val Asp Glu Lys Leu Leu 450 455 460 Phe Phe Ala Gln Gln Ala Gln Arg Arg Arg Gly Lys Ala Gly Arg Ala 465 470 475 480 Lys Asn Val Ile Phe Ser Glu Asp Arg Gly Arg Tyr Ile Lys Pro Met 485 490 495 Leu Pro Lys Gly Pro Val Lys Arg Leu Ala Val Asp Ala Thr Leu Arg 500 505 510 Ala Ala Ala Pro Tyr Gln Lys Leu Arg Arg Glu Lys Asp Thr Glu Asn 515 520 525 Arg Arg Lys Val Tyr Val Glu Lys Thr Asp Met Arg Ala Lys Arg Met 530 535 540 Ala Arg Lys Ala Gly Ala Leu Val Ile Phe Val Val Asp Ala Ser Gly 545 550 555 560 Ser Met Ala Leu Asn Arg Met Gln Asn Ala Lys Gly Ala Ala Leu Lys 565 570 575 Leu Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Ser Ile Ile Pro 580 585 590 Phe Arg Gly Asp Ser Ala Glu Val Leu Leu Pro Pro Ser Arg Ser Ile 595 600 605 Ala Met Ala Arg Lys Arg Leu Glu Arg Leu Pro Cys Gly Gly Gly Ser 610 615 620 Pro Leu Ala His Gly Leu Thr Thr Ala Val Arg Val Gly Leu Asn Ala 625 630 635 640 Glu Lys Ser Gly Asp Val Gly Arg Ile Met Ile Val Ala Ile Thr Asp 645 650 655 Gly Arg Ala Asn Ile Ser Leu Lys Arg Ser Asn Asp Pro Glu Ala Ala 660 665 670 Ala Ala Ser Asp Ala Pro Lys Pro Thr Ser Gln Glu Leu Lys Asp Glu 675 680 685 Ile Ile Glu Val Ala Ala Lys Ile Tyr Lys Thr Gly Met Ser Leu Leu 690 695 700 Val Ile Asp Thr Glu Asn Lys Phe Val Ser Thr Gly Phe Ala Lys Glu 705 710 715 720 Ile Ala Arg Val Ala Gln Gly Lys Tyr Tyr Tyr Leu Pro Asn Ala Ser 725 730 735 Asp Ala Val Val Ser Leu Ala Thr Arg Glu Ala Leu Ala Ala Leu Lys 740 745 750 Ser Ser 52 421 PRT Glycine max 52 Met Ala Ser Ala Leu Gly Thr Ser Ser Ile Ala Val Leu Pro Ser Arg 1 5 10 15 Tyr Phe Ser Ser Ser Ser Ser Lys Pro Ser Ile His Thr Leu Ser Leu 20 25 30 Thr Ser Gly Gln Asn Tyr Gly Arg Lys Phe Tyr Gly Gly Ile Gly Ile 35 40 45 His Gly Ile Lys Gly Arg Ala Gln Leu Ser Val Thr Asn Val Ala Thr 50 55 60 Glu Val Asn Ser Val Glu Gln Ala Gln Ser Ile Ala Ser Lys Glu Ser 65 70 75 80 Gln Arg Pro Val Tyr Pro Phe Ser Ala Ile Val Gly Gln Asp Glu Met 85 90 95 Lys Leu Cys Leu Leu Leu Asn Val Ile Asp Pro Lys Ile Gly Gly Val 100 105 110 Met Ile Met Gly Asp Arg Gly Thr Gly Lys Ser Thr Thr Val Arg Ser 115 120 125 Leu Val Asp Leu Leu Pro Glu Ile Lys Val Val Ala Gly Asp Pro Tyr 130 135 140 Asn Ser Asp Pro Gln Asp Pro Glu Phe Met Gly Val Glu Val Arg Glu 145 150 155 160 Arg Val Leu Gln Gly Glu Glu Leu Ser Val Val Leu Thr Lys Ile Asn 165 170 175 Met Val Asp Leu Pro Leu Gly Ala Thr Glu Asp Arg Val Cys Gly Thr 180 185 190 Ile Asp Ile Glu Lys Ala Leu Thr Glu Gly Val Lys Ala Phe Glu Pro 195 200 205 Gly Leu Leu Ala Lys Ala Asn Arg Gly Ile Leu Tyr Val Asp Glu Val 210 215 220 Asn Leu Leu Asp Asp His Leu Val Asp Val Leu Leu Asp Ser Ala Ala 225 230 235 240 Ser Gly Trp Asn Thr Val Glu Arg Glu Gly Ile Ser Ile Ser His Pro 245 250 255 Ala Arg Phe Ile Leu Ile Gly Ser Gly Asn Pro Glu Glu Gly Glu Leu 260 265 270 Arg Pro Gln Leu Leu Asp Arg Phe Gly Met His Ala Gln Val Gly Thr 275 280 285 Val Arg Asp Ala Glu Leu Arg Val Lys Ile Val Glu Glu Arg Gly Arg 290 295 300 Phe Asp Lys Asn Pro Lys Glu Phe Arg Asp Ser Tyr Lys Ala Glu Gln 305 310 315 320 Glu Lys Leu Gln Gln Gln Ile Thr Ser Ala Arg Ser Val Leu Ser Ser 325 330 335 Val Gln Ile Asp Gln Asp Leu Lys Val Lys Ile Ser Lys Val Cys Ala 340 345 350 Glu Leu Asn Val Asp Gly Leu Arg Gly Asp Ile Val Thr Asn Arg Ala 355 360 365 Ala Lys Ala Leu Ala Ala Leu Lys Gly Arg Asp Asn Val Ser Ala Glu 370 375 380 Asp Ile Ala Thr Val Ile Pro Asn Cys Leu Arg His Arg Leu Arg Lys 385 390 395 400 Asp Pro Leu Glu Ser Ile Asp Ser Gly Leu Leu Val Thr Glu Lys Phe 405 410 415 Tyr Glu Val Phe Ser 420 53 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 53 ggaagcatag catgcaaacc ac 22 54 23 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 54 cacttcaatg ggtggaagca tag 23 

What is claimed is:
 1. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having magnesium chelatase subunit CHLI activity, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:50 have at least 95% sequence identity based on the Clustal alignment method with multiple alignment default parameters of Gap Penalty=10, Gap Length Penalty=10, and pairwise alignment default parameters of Ktuple=1, Gap Penalty=3, Window=5 and Diagonals Saved=5, or (b) the complement of the nucleotide sequence, wherein the complement and the nucleotide sequence contain the same number of nucleotides and are 100% complementary.
 2. The polynucleotide of claim 1, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:49.
 3. The polynucleotide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:50.
 4. A vector comprising the polynucleotide of claim
 1. 5. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to a regulatory sequence.
 6. A method for transforming a cell comprising transforming a cell with the polynucleotide of claim
 1. 7. A cell comprising the recombinant DNA construct of claim
 5. 8. A method for producing a plant comprising transforming a plant cell with the polynucleotide of claim 1 and regenerating a plant from the transformed plant cell.
 9. A plant comprising the recombinant DNA construct of claim
 5. 10. A seed comprising the recombinant DNA construct of claim
 5. 